In this study, we found significant sex differences in thrombosis in mice; these differences were abrogated in mice deficient in GH. Specific GH administration patterns modulated changes in expression of the genes encoding the principal regulators of thrombin generation in plasma, protein C (Proc), AT (Serpinc1), and HCII (Serpind1) and the purported procoagulant inhibitor PCI (Serpina5) in the liver. Thus, the changes in whole-blood clotting and PE susceptibility appear to be mediated by the effects of GH and resultant changes in expression of several plasma-based modulators of thrombin generation.
To our knowledge, this represents the first description of the effects of GH on sex differences in coagulation or thrombosis in humans or rodents. We believe this is an important step in understanding the biological mechanism underlying important sex differences in disease susceptibility. While there are potential confounding effects of age, supplemental sex hormones, or pregnancy, and while there is no evidence of sex differences in the rate of incident VTE (44
), there is substantial evidence for strong sex differences in the rate of recurrent VTE (1
). Thus, there is great value in understanding the mechanisms underlying such differences, as they may lead to novel paradigms of diagnosis or management of thrombotic disorders.
There is also substantial evidence of sex-specific effects in thrombosis in mouse models. Such models are of fundamental importance to the field of hemostasis and thrombosis. We and others have found that male mice are more susceptible to thrombosis in vivo, yet there has been, until now, no explanation for such differences (11
). We hope that this work will help to elucidate the very real sex differences in thrombosis models in mice and may explain why effects of drugs or gene knockout studies have been sex dependent.
To our knowledge, our study represents the most detailed analysis of the effects of GH on coagulation yet published and is the first to examine the effects of GH on thrombosis in vivo. Previous studies have evaluated the effect of GH on coagulation in rats (45
). However, these studies demonstrated changes exclusively in either male or female rats and found conflicting results, and none measured effects on thrombosis in vivo. The data regarding the effects on GH on human thrombosis are sparse. A current review of the literature reveals 1 recent article examining the effect of GH replacement on coagulation parameters in male and female patients with GH deficiency. There were no differences between control and GH-deficient patients, but replacement of GH caused prolongation of aPTT only in males and of the prothrombin time only in females (47
The effects of GH on thrombosis in this study were modulated at least to some degree by changes in the concentration of anticoagulant inhibitors. Each of these inhibitors is known to be a principal regulator of thrombin generation in plasma. The models used in this study are clearly thrombin dependent. We previously demonstrated that mice with abnormal platelet thrombin signaling are protected in the well-characterized PE model (11
). Here, we showed that the model is thrombosis dependent, with dose-dependent changes in intravascular thrombus formation and subsequent decreases in postmortem lung perfusion (11
). APC has also been shown to protect mice in a similar model of thrombin-induced PE (48
), and in prior work, we have found that mice with heterozygous deficiency of AT are more susceptible in the PE model, with median survival times of 210 versus 280 seconds in AT+/–
male mice (R.E. Levy and E.J. Weiss, unpublished observations). Indeed, while a direct comparison across experiments is limited by such caveats as background strain and differences in experimental conditions, the magnitude of protection afforded by GH deficiency was at least a great as previously observed in Par3
- and Par4
-deficient animals. Triggering thrombin generation and thrombosis with dilute TF as we have done in our models incorporates both extrinsic and intrinsic pathway processes. This model is therefore sensitive to alterations in concentration or activity of primary inhibitors of thrombin generation or thrombin’s actions; or those of inhibitors of thrombin-mediated amplification. Alterations in concentrations of any of these molecules would alter the dynamics of thrombin generation or the activity of thrombin and would certainly change the rate of thrombus formation in vitro and in vivo (49
It is important to note 2 important caveats about the PE model: (a) it is weight based; therefore, male mice do receive a higher dose of thromboplastin than females. Furthermore, the male-female weight difference is largely abrogated in lit mice. While we cannot rule out an effect of weight on mortality in this specific line, we have previously characterized several other mouse knockout lines and inbred strains and have observed no relationship between weight and thromboplastin susceptibility (R.E. Levy and E.J. Weiss, unpublished observations). (b) As described above, both the PE model and the whole-blood clotting model could be influenced by changes in platelet number or function. While we cannot rule out a platelet contribution, we feel that the differences in clotting in PPP, in the coagulation factor assays, and in the APC assays (all performed on plasma) argue for a significant effect of plasma-based proteins.
The effects on the protein C pathway appear to be dual. The gene expression data suggest that there are GH-mediated sex differences in the synthesis of Proc
. In addition, the observation of acquired APC resistance strongly suggests that there are also changes in concentration or activity of one or more of the known APC cofactors such as protein S (50
) or growth arrest–specific gene 6 (51
) or known APC antagonists such as PCI (Serpina5
), α1-proteinase inhibitor, or α2-macroglobulin (52
). Among the genes we tested, both the baseline sex differences and the effects of GH were greatest on Serpina5
is expressed abundantly in human liver but is not expressed at significant levels outside of the reproductive tract in rodents (53
). The mouse knockout of Serpina5
offered no insight into its role in thrombosis because of the low baseline expression (53
). To our knowledge, mice with a targeted disruption in Serpina5
have not been examined for changes in APC resistance, thrombin generation, or thrombosis in vivo. Given that GH caused a dose-dependent increase in APC resistance while causing a concomitant increase in the expression of Serpina5
, it is tempting to implicate Serpina5
in the GH-mediated changes in thrombosis. However, the true effect of Serpina5
on thrombosis in vivo remains to be determined.
Work in the past 20 years has helped uncover the molecular basis for how sex-specific patterns of GH secretion lead to sex differences in hepatic gene expression and effects on growth and metabolism. The seminal work of Norstedt and Palmiter established that sex-specific patterns of GH release can mediate sex-specific patterns of liver protein expression (23
). They hypothesized that male-specific liver proteins were induced by the longer male GH interpulse interval and subsequent discontinuous IGF-1 secretion. Female mice — by virtue of the continuous presence of GH — have continuous IGF-1 production and therefore downregulation of IGF-1 receptors, which induce female-specific proteins. lit
mice — with near absent GH — have markedly decreased IGF-1 production and therefore a female-like pattern of liver gene expression (23
). Since then, there has been tremendous progress in understanding the signaling pathways involved in mediating sex-specific liver gene expression. Much of this work has focused on mice deficient in STAT5 factors. STAT5-knockout mice are GH resistant and have demonstrated significant changes in sex-specific gene expression patterns (29
). The IGF-1 hypothesis is bolstered by work in STAT5-deficient animals, as the loss of STAT5 generally feminizes liver gene expression, and STAT5 is necessary for normal IGF-1 production in the liver (56
). However, by virtue of the loss of IGF-1–mediated feedback inhibition, STAT5-deficient animals have supraphysiologic GH levels (29
). Therefore, the specific effects of decreased IGF-1 versus increased GH itself remain to be determined.
We think that this work raises interesting and unresolved questions. Most notably, Why are there sex differences in thrombosis or thrombosis-related gene expression? Recently, it was shown that Par4
deficiency rescues fetal loss in a genetic model exploring the interactions of fetal and maternal thrombophilia (58
). This and other work suggest that increases in maternal thrombin generation are deleterious to fetal development and may partially explain the relative decrease in baseline clotting in females. While this and other questions remain unanswered, with this work, we believe that we have described a novel and important mechanism driving sex differences in clotting in mice that ultimately may help to explain the strong sex differences in susceptibility to human clotting-related diseases. We hope that this work and future work designed to define the mechanism of these important sex differences might someday lead to advances in sex-specific risk assessment, diagnosis, or management of thrombosis-related diseases.