Ethan J. Weiss

A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis

BACKGROUND Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggregation. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, PlA2, of the gene encoding glycoprotein IIIa in kindreds with a high prevalence of premature myocardial infarction. METHODS To investigate the relation between the PlA2 polymorphism and acute coronary syndromes, we conducted a case-control study of 71 case patients with myocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the PlA genotype: reverse dot blot hybridization and allele-specific restriction digestion. RESULTS The prevalence of PlA2 was 2.1 times higher among the case patients than among the controls (39.4 percent vs. 19.1 percent, P=0.01). In a subgroup of patients whose disease began before the age of 60 years, the prevalence of PlA2 was 50 percent, a value that was 3.6 times that among control subjects under 60 years of age (13.9 percent, P=0.002). Among subjects with the PlA2 polymorphism, the odds ratio for having a coronary event was 2.8 (95 percent confidence interval, 1.2 to 6.4). In the patients less than 60 years of age at the onset of disease, the odds ratio was 6.2 (95 percent confidence interval, 1.8 to 22.4). CONCLUSIONS We observed a strong association between the PlA2 polymorphism of the glycoprotein IIIa gene and acute coronary thrombosis, and this association was strongest in patients who had had coronary events before the age of 60 years.

PAR3 is a cofactor for PAR4 activation by thrombin

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptors acting as an accessory molecule to present ligand to another receptor.

Role of thrombin signalling in platelets in haemostasis and thrombosis

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

Protease-activated Receptors 1 and 4 Are Shut Off with Distinct Kinetics after Activation by Thrombin

Protease-activated receptors 1 and 4 (PAR1 and PAR4) mediate thrombin signaling in human platelets. Whether these receptors are redundant, interact, or serve only partially overlapping functions is unknown. We report that PAR1 and PAR4 signal with distinct tempos. In transfected fibroblasts, PAR4 triggered substantially more phosphoinositide hydrolysis per activated receptor than PAR1 and was shut off more slowly than PAR1. Shutoff and internalization of PAR1 depends upon phosphorylation of its carboxyl tail upon receptor activation. In contrast to PAR1, phosphorylation of PAR4 was undetectable, and activation-dependent internalization of PAR4 was much slower than that seen for PAR1. Mutation of potential phosphorylation sites in the carboxyl tail of PAR1 enhanced PAR1 signaling, whereas analogous mutations in PAR4 had no effect. Thus PAR4 signaling is shut off less rapidly than PAR1, probably due to differences in receptor phosphorylation. PAR1 and PAR4 also signaled with distinct tempos in platelets. PAR1 triggered a rapid and transient increase in intracellular calcium, whereas PAR4 triggered a more prolonged response. Together, the tempo of these responses accounted for that triggered by thrombin. Thus differences in the rates at which PAR1 and PAR4 are shut off allow thrombin to trigger intracellular signaling with distinct temporal characteristics.

Abrogation of growth hormone secretion rescues fatty liver in mice with hepatocyte-specific deletion of JAK2

Non-alcoholic fatty liver disease is associated with multiple comorbid conditions, including diabetes, obesity, infection, and malnutrition. Mice with hepatocyte-specific disruption of growth hormone (GH) signaling develop fatty liver (FL), although the precise mechanism underlying this finding remains unknown. Because GH signals through JAK2, we developed mice bearing hepatocyte-specific deletion of JAK2 (referred to herein as JAK2L mice). These mice were lean, but displayed markedly elevated levels of GH, liver triglycerides (TGs), and plasma FFAs. Because GH is known to promote lipolysis, we crossed GH-deficient little mice to JAK2L mice, and this rescued the FL phenotype. Expression of the fatty acid transporter CD36 was dramatically increased in livers of JAK2L mice, as was expression of Pparg. Since GH signaling represses PPARγ expression and Cd36 is a known transcriptional target of PPARγ, we treated JAK2L mice with the PPARγ-specific antagonist GW9662. This resulted in reduced expression of liver Cd36 and decreased liver TG content. These results provide a mechanism for the FL observed in mice with liver-specific disruption in GH signaling and suggest that the development of FL depends on both GH-dependent increases in plasma FFA and increased hepatic uptake of FFA, likely mediated by increased expression of CD36.

Hepatocyte-Specific Disruption of CD36 Attenuates Fatty Liver and Improves Insulin Sensitivity in HFD-Fed Mice

CD36/FAT (fatty acid translocase) is associated with human and murine nonalcoholic fatty liver disease, but it has been unclear whether it is simply a marker or whether it directly contributes to disease pathogenesis. Mice with hepatocyte-specific deletion of Janus kinase 2 (JAK2L mice) have increased circulating free fatty acids (FAs), dramatically increased hepatic CD36 expression and profound fatty liver. To investigate the role of elevated CD36 in the development of fatty liver, we studied two models of hepatic steatosis, a genetic model (JAK2L mice) and a high-fat diet (HFD)-induced steatosis model. We deleted Cd36 specifically in hepatocytes of JAK2L mice to generate double knockouts and from wild-type mice to generate CD36L single-knockout mice. Hepatic Cd36 disruption in JAK2L livers significantly improved steatosis by lowering triglyceride, diacylglycerol, and cholesterol ester content. The largest differences in liver triglycerides were in species comprised of oleic acid (C18:1). Reduction in liver lipids correlated with an improvement in the inflammatory markers that were elevated in JAK2L mice, namely aspartate aminotransferase and alanine transaminase. Cd36 deletion in mice on HFD (CD36L-HFD) reduced liver lipid content and decreased hepatic 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-FA uptake as compared with CON-HFD. Additionally, CD36L-HFD mice had improved whole-body insulin sensitivity and reduced liver and serum inflammatory markers. Therefore, CD36 directly contributes to development of fatty liver under conditions of elevated free FAs by modulating the rate of FA uptake by hepatocytes. In HFD-fed animals, disruption of hepatic Cd36 protects against associated systemic inflammation and insulin resistance.

Sex differences in thrombosis in mice are mediated by sex-specific growth hormone secretion patterns

Sex differences in thrombosis are well described, but their underlying mechanism(s) are not completely understood. Coagulation proteins are synthesized in the liver, and liver gene expression is sex specific and depends on sex differences in growth hormone (GH) secretion--males secrete GH in a pulsatile fashion, while females secrete GH continuously. Accordingly, we tested the hypothesis that sex-specific GH secretion patterns cause sex differences in thrombosis. Male mice were more susceptible to thrombosis than females in the thromboplastin-induced pulmonary embolism model and showed shorter clotting times ex vivo. GH-deficient little (lit) mice were protected from thrombosis, and pulsatile GH given to lit mice restored the male clotting phenotype. Moreover, pulsatile GH administration resulted in a male clotting phenotype in control female mice, while continuous GH caused a female clotting phenotype in control male mice. Expression of the coagulation inhibitors Proc, Serpinc1, Serpind1, and Serpina5 were strongly modulated by sex-specific GH patterns, and GH modulated resistance to activated protein C. These results reveal what we believe to be a novel mechanism whereby sex-specific GH patterns mediate sex differences in thrombosis through coordinated changes in the expression of coagulation inhibitor genes in the liver.

Higher Prevalence of GPIIIa PlA2 Polymorphism in Siblings of Patients With Premature Coronary Heart Disease

BACKGROUND The Pl(A2) polymorphism of GPIIIa has been associated with unstable coronary syndromes in some studies, but the association has remained debated. None of the previous studies have focused on families at high risk. Risk factors tend to cluster within kindreds with high prevalence of premature coronary heart disease (CHD). Therefore, a heightened prevalence of the Pl(A2) polymorphism among siblings of patients with CHD would support the hypothesis that Pl(A2) is linked, directly or indirectly, to CHD. OBJECTIVES To measure the prevalence of the Pl(A2) polymorphism among siblings of patients with CHD before the age of 60 years and to seek an association between the Pl(A2) polymorphism and established atherosclerotic and thrombogenic risk factors. METHODS From January 1994 to April 1996, we genotyped 116 asymptomatic siblings (60 Caucasians, 56 Afro-Caribbeans) of patients with CHD manifestations before the age of 60 years for the Pl(A) polymorphism (also called HPA-1). A control cohort was used for comparison, consisting of individuals that were matched for race and geographic area but were free of CHD (n = 268, 168 Caucasians and 100 Afro-Caribbeans). In addition, we have characterized the sibling cohort for other atherogenic and thrombogenic risk factors. RESULTS The prevalence of Pl(A2)-positive individuals (Pl(A2)[+], Pl(A1/A2) heterozygotes plus Pl(A2/A2) homozygotes) in the sibling cohort was high: 41.4%. When analyzed separately, the prevalence of Pl(A2)(+) siblings was 53.3% among Caucasians and 28.6% among Afro-Caribbeans. There was no association between Pl(A2) and other established atherogenic or thrombogenic risk factors. Interestingly, the clustering of other risk factors was lesser among Pl(A2)(+) siblings than their Pl(A1) counterparts. CONCLUSIONS This study supports the hypothesis that the prevalence of Pl(A2)(+) individuals is high in kindreds with premature CHD. Hence, like the established risk factors that tend to cluster in families with premature CHD and contribute strongly to the accelerated atherosclerotic process affecting these individuals, the Pl(A2) polymorphism of GPIIIa may represent an inherited risk that promotes the thromboembolic complications of CHD. That these asymptomatic Pl(A2)(+) siblings had overall less established risk factors than their Pl(A1) counterparts might represent an explanation for why they remained asymptomatic despite their Pl(A2) positivity.

Disruption of JAK2 in adipocytes impairs lipolysis and improves fatty liver in mice with elevated GH.

Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic expression of the metabolic syndrome, and its prevalence is increasing. The factors that influence the development of fatty liver and its progression to steatohepatitis and cirrhosis are not well understood. The pleiotropic hormone, GH, has been associated with an increased risk of NAFLD in humans and mice. GH is known to have diverse effects on lipid metabolism including decreasing body fat in vivo, presumably through stimulation of lipolysis via an undefined mechanism. Previously we described mice with hepatocyte-specific deletion of the GH signaling mediator, Janus kinase 2 (JAK2L). JAK2L animals have elevated serum GH, reduced body fat, high liver triglyceride content, and increased serum markers of hepatocyte injury (alanine transaminase and aspartate transaminase). We aimed to determine whether the elevation of GH in JAK2L mice contributed to fatty liver by promoting lipolysis directly in adipocytes. We generated mice with adipocyte-specific disruption of JAK2 (JAK2A) and found that GH resistance in adipocytes reduced lipolysis and increased body fat. JAK2A mice were then crossed to JAK2L mice, and the resultant JAK2L/A animals had increased body fat and decreased lipolysis, despite elevated circulating GH. Furthermore, the increased triglyceride content, serum alanine transaminase, and serum aspartate transaminase observed in JAK2L mice were nearly normalized with the additional disruption of JAK2 in adipocytes (JAK2L/A mice). Our results offer novel mechanistic insights into the long-recognized effects of GH on lipid flux and suggest that GH signaling may play an important regulatory role in the development of NAFLD.

HIV Infection Is Associated With Decreased Thrombin Generation

BACKGROUND Excess risk of cardiovascular disease occurs in effectively treated individuals with human immunodeficiency virus (HIV) infection. Although elevated plasma D-dimer levels are associated with increased morbidity and mortality, the impact of HIV infection on coagulation in vivo has not been well studied. METHODS We measured D-dimers, antithrombin, endogenous thrombin potential (ETP; a functional measure of thrombin generation in vitro), thrombin/antithrombin complexes (TAT; a measure of thrombin generation in vivo), tissue factor, prothrombin fragment 1 + 2 (F1+2), and normalized APC sensitivity ratio (nAPCsr) in 199 HIV-positive men who were receiving antiretroviral therapy and had an undetectable HIV RNA level, in 79 HIV-positive untreated men, and in 39 uninfected controls. RESULTS Median antithrombin levels were higher while the ETP was lower among HIV-infected adults (treated and untreated), compared with controls. There were few differences between coagulation markers in the 2 HIV groups. Compared with controls, the nAPCsr was lower in treated men and the TAT level was lower in untreated individuals. We observed little difference among measured levels of D-dimer, tissue factor, or F1+2 between HIV-infected individuals and controls. Antiretroviral therapy exposure was associated with a lower antithrombin level, a lower nAPCsr, and a lower ETP, while history of opportunistic infection was associated with a higher nAPCsr. CONCLUSIONS HIV infection is associated with decreased thrombin generation, as measured by the ETP, and an increased antithrombin level. These data suggest that HIV infection may not be associated with increased propensity toward clotting, as has been suggested on the basis of isolated measures of D-dimer levels.