This chapter summarizes what is known about normal and abnormal coagulation of blood in inbred mice. Until 1960 mouse blood had not been studied with respect to the amounts of various clotting factors present normally in healthy mice and absent in a number of hemorrhagic syndromes.
NORMAL BLOOD CLOTTING
Methods of assaying normal mouse blood for clotting factors and for the amounts of each component necessary for normal clotting are given by Meier et al. ( 1960, 1962) and Meier ( 1963). Knowledge of clotting factors extracted from mouse blood is of value in (1) the diagnosis of abnormal clotting in man, (2) the study of the mechanism of clotting as a biological process, and (3) the assay of certain liver functions. Blood coagulation is a highly complicated biological phenomenon. A tentative and oversimplified scheme of the clotting mechanism is shown in Figure 18-1. It is presented to familiarize the reader with the various synonyms in common use and to provide some understanding of the different factorial interactions. A separate assay is available for the three stages, each consisting of several linked reactions.
ABNORMAL BLOOD CLOTTING
Several disorders of the clotting mechanism including both excessive bleeding and hypercoagulability have been described in inbred mice.
Stuart-Prower factor deficiency
A spontaneous bleeding or hemorrhagic diathesis occurred in certain inbred strains of mice at The Jackson Laboratory in 1960 ( Meier et al., 1962). About 80 per cent of males of the SWR/J strain and about 20 per cent of males of the DBA/2J strain died with bilateral hemothorax and sometimes with multiple bleedings elsewhere in the body, either subcutaneous, perivesicular, or subdural. The mice were anemic and jaundiced. The major histopathological findings were myocarditis and various degrees of hepatomegaly. Elevated lactic dehydrogenase was associated with myocarditis. Anemia and icterus seemed to be consequences f extensive hemorrhage rather than of hepatic dysfunction since alkaline and acid serumphosphatase and glutamic-oxalic transaminase were, except for lowered serum glutamic-pyruvic transaminase, within normal range or only slightly altered.
The affected mice had single or multiple "prothrombin complex" deficiencies, including deficiencies of the Stuart-Prower factor or factor X, plasma thromboplastin component (PTC) or factor IX, serum prothrombin conversion factor or factor VIII, and prothrombin. Activities of the first three factors, determined by specific assays using both human and normal mouse blood were less than 3 per cent of normal and the last was less than 10 per cent. Accelerator globulin (Ac-G or factor V), Hageman factor, and fibrinogen were normal. The clotting abnormality which first appeared in the course of the disease was always Stuart-Prower factor deficiency.
Two features were particularly noteworthy: (1) occurrence only in males of two strains and (2) consistency of the lesions both microscopically and biochemically. Although myocarditis was the most severe histological alteration, liver dysfunction with respect to clotting factor(s) was the most important clinical consequence, leading eventually to death from loss of blood. The simultaneous occurrence of the condition in two strains suggested that the disease was due to an environmental agent rather than to a mutation and that these strains differed from the others in their genetic susceptibility. A search for possible agents implicated ethylene oxide sterilization of wood shavings used as mouse-cage bedding. The presence of one or more glycols in shavings gassed with ethylene oxide was shown chromatographically. Dilute solutions of ethylene glycol, administered by gavage, eliminated or greatly degreased the activity of factor X in less than 72 hours ( Allen et al., 1962). In addition, we had observed the spontaneous factor X deficiency only in those bleeding mice that had prolonged contact with sterilized bedding. Mice not only touch shavings with parts of their bodies, but continually chew them. Discontinuing gas sterilization eliminated the hemorrhagic disease.
No explanation is available for the peculiar strain and sex distribution, other than the obvious genetic differences in response to a toxic agent.
Factor VII deficiency
Clotting factor determinations in mice have been found to be extremely sensitive indicators of liver function. For example, clotting abnormalities associated with liver damage caused by a variety of bacterial diseases and toxicities have appeared before other functional deficiencies of the liver were detected. An example is factor X deficiency induced by ethylene glycol. However, liver damage usually affects several clotting factors. For instance, hypoprothrombinemia is usually most severe in chloroform poisoning, but the accelerator globulin (factor V) is also greatly diminished.
Of all clotting factors, a factor VII deficiency occurs most often in association with impaired liver function. It readily results from hypovitaminosis K in most animal species including man ( Alexander et al., 1959). However, in mice, a diet without any vitamin K complex did not induce a factor VII deficiency. Coupled with other evidence this suggests that under ordinary conditions intestinal microorganisms are able to supply much, if not all, of the required vitamin K.
Signs of vitamin K deficiency in female mice may be induced by dl-γ-tocopherol quinone, a vitamin K and E analogue ( Woolley, 1945). Hemorrhages presumably due to factor VII deficiency occur only in the reproductive system of pregnant mice. The compound is without effect in males and nonpregnant females. However, pregnant females that received daily oral doses of 100 mg of the quinone had prothrombin times similar to the controls. Feeding of 3,3-methylene-bis-4-hydroxycoumarin, another antimetabolite of vitamin K, caused some signs of the vitamin K deficiency but did not produce resorption of fetuses and vaginal hemorrhages in pregnant mice ( Woolley, 1945).
Left auricular thrombosis
Left auricular thrombosis occurs in high incidence (66 per cent) among older breeding females or inactive breeders of the BALB/cJ strain. The significance of repeated pregnancies rather than age alone is indicated by the fact that BALB/cJ males and virgin or ovariectomized females never develop the condition. Although repeated pregnancies in females of most strains do not affect coagulation or only irregularly cause some reduction in certain precoagulant factors, BALB/cJ mice suffer from severe deficiencies of PTC or factor IX (about 40 per cent of normal), antihemophilic factor (AHF) or factor XIII (about 60 per cent of normal), Stuart-Prower factor or factor X (about 50 per cent of normal), and prothrombin (about 33 per cent of normal). These deficiencies occur shortly before parturition. Hageman factor and Ac-G are always normal. These prothrombin-complex abnormalities disappear a few days after birth; plasma prothrombin rebound may be 20 to 25 per cent above normal within 2 days of parturition. The rebound phases are only moderately or not at all reflected in the "one-stage" prothrombin time. Attempts to measure fibrinolytic activity in blood from thrombotic mice fail because of the very low activity found normally in mouse blood ( Meier et al., 1960).
We believe that the postpartum prothrombin-rebound, similar to that observed in man following Dicumerol and heparin therapy, may have clinical implications. The thrombus probably forms rather quickly and is primary, whereas endocardial damage is probably secondary. The clinical condition associated with advanced stages of auricular thrombosis consists of unthriftiness, labored breathing from lung congestion, and usually subcutaneous edema. At necropsy the left auricle is three to five times the normal size. Since the disease can be diagnosed readily or at least suspected in breeders pregnant more than six times, it will be useful in studies of thrombosis formation, lysis, and potential fibrinolytic compounds ( Meier and Hoag, 1962).
A similar condition can be induced in young adult mice of a number of strains by feeding a hyperlipotropic diet containing 28 per cent fat as lard and 8 per cent protein as casein. After about 7 weeks on this diet the mice develop atrial necrosis, resulting in formation of mural thrombi and reaching critical or terminal dimensions in 10 to 12 weeks. Betaine (hydrochloride, 2 mg/100 g of diet) does not prevent the lesions ( Ball, 1962).
We have referred to the pertinent literature on aspects of normal blood clotting in mice. The papers cited give the methods for factorial assays and the normal values of all factors involved in coagulation. A simplified scheme of the factors concerned in blood coagulation has been presented. The examples of single or multiple factor deficiency syndromes discussed include the Stuart-Prower (factor X), factors VII and VIII, and the plasma thromboplastin component (factor IX). Each deficiency depends upon a particular genetic susceptibility whether or not it occurs spontaneously or is induced.
1The writing of this chapter was supported in part by the National Hemophilia Foundation and in part by Public Health Service Grant CA 04691 from the National Cancer Institute.
Alexander, B., A. Kilma, R. Coleman, E. Scholtz, and A. DiFrancesco. 1959. New "hemophiloid" defects: some clinico-laboratory and experimental abnormalities in thromboplastin generation, p. 137-157. In K.M. Brinkhous and P. DeNichola [ed.] Hemophilia and Other Hemorrhagic States. University of North Carolina Press, Chapel Hill.
Allen, R.C., H. Meier, and W.C. Hoag. 1962.
Ethylene glycol produced by ethylene oxide sterilization and its effect on blood-clotting factors in an inbred strain of mice.
Nature 193: 387-388.
See also PubMed.
Ball, C.T. 1962. Cardiac lesions in mice fed high fat, low protein diets. Anat. Rec. 142: 212.
Meier, H. 1963. Experimental Pharmacogenetics. Academic Press, New York. 213 p.
Meier, H., R.C. Allen, and W.G. Hoag. 1960.
Normal blood clotting of inbred mice.
Amer. J. Physiol. 201: 375-378.
See also PubMed.
Meier, H., R.C. Allen, and W.G. Hoag. 1962.
Spontaneous hemorrhagic diathesis in inbred mice due to single or multiple "prothrombin complex" deficiencies.
Blood 19: 501-514.
See also MGI.
Meier, H., and W.G. Hoag. 1962.
Studies on left auricular thrombosis in mice.
Exp. Med. Surg. 19: 317-322.
See also PubMed.
Woolley, D.W. 1945. Some biological effects produced by α-tocopherol quinone. J. Biol. Chem. 159: 59-66.