Defined as estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m², chronic kidney disease (CKD) stage 3 to 5 1 is an established risk factor for cardiovascular morbidity and mortality 23. This association begins in the pre-clinical stages of kidney disease, when kidney function may be better estimated by cystatin C (eGFRcys) 456. The mechanisms underlying the association of CKD with cardiovascular morbidity and mortality are inadequately understood. In multiple cohorts with and without CKD, the associations of lower eGFR with higher cardiovascular risk persist despite adjustment for traditional risk factors and inflammation 78910, prompting investigation into novel pathways between renal and cardiovascular disease (CVD).

Atherosclerosis represents a pernicious interplay between systems of inflammation and coagulation 11. Multiple investigators have reported an association between creatinine-based CKD and elevated levels of inflammatory and thrombotic markers 1213. In studies from the Health, Aging and Body Composition Study and the Multi-Ethnic Study of Atherosclerosis (MESA), subjects with mildly reduced kidney function measured with cystatin C had only modest elevations in inflammatory markers 13141516. Furthermore, the positive association of cystatin C with cardiovascular and overall mortality is only partially attenuated by adjustment for C-reactive protein (CRP), interleukin-6 (IL-6) or TNF-α 56. However, whether or not dysregulation of hemostasis contributes to the association of CKD and CVD has not been well evaluated.

We designed these analyses to explore the associations of reduced kidney function, as measured by eGFRcys and CKD Epidemiology Collaboration (eGFRcr), with hemostatic factors in an ethnically diverse cohort free of clinical cardiovascular disease.

In a large, multiethnic cohort free of cardiovascular disease and with a spectrum of kidney function ranging from normal to moderate CKD, even small decrements in eGFR were associated with significantly higher levels of sTM, sTF, D-Dimer, vWF, factor VIII, PAP and TFPI. Our results suggest that dysregulation of hemostasis could play an important pathologic role in CKD. In particular, these associations may help to explain the consistently strong and independent associations of cystatin C with cardiovascular and overall mortality, despite adjustment for known risk factors including CRP 5, IL-6 and TNF-α 6.

Soluble thrombomodulin and soluble tissue factor had the largest associations with eGFRcys. sTM is a receptor for thrombin found on vascular and lymphatic endothelial cells. Injured endothelial cells release sTM into the circulation, where it binds thrombin, inhibiting it from forming fibrin and activating platelets. sTM has additional anti-thrombotic activity as a cofactor for the thrombin-catalyzed activation of protein C 23. Elevated sTM is associated with lower eGFR in CKD and end stage renal disease ESRD 2425, and our results extend these findings to a broader spectrum of eGFR. The association of sTM and cardiovascular disease varies by patient population: in healthy individuals, lower sTM predicts coronary heart disease (CHD), but in patients with established CHD, higher sTM levels are associated with recurrence of CHD and with higher mortality 26. Whether higher levels of sTM are associated with CVD or mortality in our cohort would require further investigation.

Tissue factor (TF) initiates the extrinsic coagulation cascade. Smooth muscle cells, adventitial fibroblasts, and pericytes constitutively express TF, but endothelial cells express TF only after injury 27. TF has a prominent role in atherosclerotic plaque rupture and acute coronary syndrome (ACS). TF is concentrated in the core of the atherosclerotic plaque; when the plaque ruptures, TF comes into contact with blood, binds factor VII and initiates the extrinsic coagulation cascade 28. Higher levels of sTF were reported previously in CKD as compared to healthy subjects 29. Epidemiological studies of sTF and CVD occurrence in healthy individuals are lacking, but in patients with ACS, high levels of sTF on admission are associated with poor outcome 30. To our knowledge this paper is the first to report an association between higher sTF and lower eGFR in patients without CKD. Whether high levels of sTF contribute to the high cardiovascular mortality of persons with kidney disease is unknown.

D-Dimer, vWF, Factor VIII, and PAP showed moderate associations with lower eGFRcys. Elevated levels of D-Dimer are associated with lower eGFR 12 and CVD 31 across the spectrum of eGFR. vWF is increased in subjects with CKD 32, and it is considered to be a marker for endothelial dysfunction 33 and CVD risk 3435. Factor VIII is increased in subjects with CKD 1232. In the Atherosclerosis Risk in Communities (ARIC) Study, factor VIII was associated with progression of CKD and with fatal MI 36. In MESA, Factor VIII was associated with overall but not cardiovascular mortality 37. Plasmin, an enzyme that increases fibrinolysis, is inactivated when complexed with a2-antiplasmin to form PAP 11. Subjects with CKD have higher levels of PAP than subjects without CKD 12. In a cohort with CAD, PAP was associated with higher CVD risk 38; in the Cardiovascular Health Study (CHS), PAP was associated with incident myocardial infarction 39.

Several of the hemostatic markers evaluated in this study are strong risk factors for venous thomboembolic disease, including D-dimer, factor VIII, and vWF 40. Recent epidemiological studies have linked CKD with risk of venous thrombosis 4142. Thus, our findings may be applicable to both arterial and venous thrombosis, and these associations may extend into higher levels of kidney function than previously appreciated.

There are several possible mechanisms to explain the association of lower eGFR and higher levels of hemostatic factors. A direct effect of decreased renal clearance may explain an increase in levels of smaller molecular weight hemostatic markers such as sTF and sTM, as these may be filtered at the glomerulus. Fragments of sTM ranging from 28 to 108 kDa can be detected in blood and urine 43. Soluble tissue factor has a MW of 29 kDa. Urinary tissue factor, with MW of 34 kDa, is elevated in glomerulonephritis and various malignancies, but it is thought to originate from the kidneys, rather than being blood borne and filtered at the glomerulus 44. Whether sTF and sTM are filtered at the glomerulus would have to be tested in animal studies. Given the higher molecular weight of D-Dimer, vWF, factor VIII and PAP, it seems unlikely that these markers are elevated as a direct result of decreased renal clearance. It is possible that elevations in these markers relate to processes initiated by smaller molecules. Conversely, kidney dysfunction may generate a thrombotic milieu indirectly through electrolyte or acid-base abnormalities, which may alter activities of enzymes involved in coagulation. Inflammation associated with lower eGFR may cause elevations in markers that are acute phase reactants. Alternatively, microvascular disease may promote the development of CKD, and in parallel, may cause elevations in these hemostatic biomarkers; in this scenario, the markers would be neither cause nor effect of lower eGFR.

A strength of this study is the unique setting, as MESA is a large, well-characterized cohort including four major race/ethnic groups. The use of cystatin C to measure eGFR increases our ability to detect associations of biomarkers across a broader spectrum of kidney function. However, there are important limitations to our study. As is typical for most large epidemiological studies, we did not use a gold standard measure of eGFR, such as iothalamate or iohexol clearance. Due to the cross-sectional design, we cannot ascertain the direction of association; it is equally possible that increased levels of thrombotic markers causes decreased eGFR, or that decreased eGFR causes the elevated markers. Finally, residual confounding may exist in our analysis, which may account for some of our findings, but would be less likely to account for the strong associations of eGFRcys with sTM and sTF.

In summary, lower eGFR was significantly associated with higher levels of sTM, sTF, D-Dimer, PAP, Factor VIII, TFPI, vWF and fibrinogen in subjects with and without CKD. Increased levels of smaller molecular weight molecules such as sTM and sTF may be a direct result of decreased renal clearance. Elevations of larger molecules may represent increased fibrinolytic activity and higher clot burden of a thrombotic milieu induced by declining kidney function. Alternatively, the atherosclerotic process may differ fundamentally in persons with lower eGFR. Further investigations may lead to a better understanding of the mechanisms underlying these associations, as well as the role of these associations in predicting cardiovascular disease in subjects with elevated cystatin C.

The authors declare that they have no competing interests.

RD drafted and revised the paper, and gave input into statistical analysis. MC helped to draft the manuscript and provided advice on hemostatic factors. AF helped draft the manuscript and provided advice on interpreting the data. LF helped draft the manuscript and provided advice on interpreting the data. WP helped draft the manuscript and provided advice on interpreting the data. CP helped draft the manuscript and provided advice on interpreting the data. CW performed statistical analysis. MS sponsored and conceived of the project, helped revise the paper, and gave input into statistical analysis. All authors read and approved the final manuscript.