Thrombin Generation Among Sudanese Essential Hypertensive Patients. KEYWORDS: Hypertension, Thrombosis, Hemostasis, Thrombin generation

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1 Thrombin Generation Among Sudanese Essential Hypertensive Patients Fathelrahman M. H 1, M. M. Hamid 2 and A. E. Mohamed 1 1 College of Medical Laboratory Science, Sudan University of Science and Technology, Sudan 2 Faculty of Medicine, University of Khartoum. ABSTRACT: This is a descriptive, prospective analytical case-control based study conducted in Khartoum State Teaching Hospitals during the period of October 2003 to January 2008 to determine the thrombin generation among Sudanese hypertensive patients. Two hundred patients (200) and fifty normal controls () were studied. The results showed a significant correlation between hypertension and family history of patients and non significant correlation between family history of hypertension and the gender of the study group. Most hypertensive patients in the study group were presented with a duration range from 1 month up to 5 years using antihypertensive drugs as a treatment. The results showed a non significant difference between the mean level of patients and controls in prothrombin fragment 1+2 (F1+2) (p=0.925) and thrombin antithrombin complex (TAT) (p=0.867). Prothrombin fragment 1+2 level (p= 0.012) was significantly correlated to severity of hypertension (stage II), while thrombin antithrombin complex level did not show significant (p=0.124) correlation to the severity of the disease. Both circulating markers of thrombin generation (Prothrombin fragment 1+2 and thrombin antithrombin complex) were significantly (p=0.000) correlated to the duration of hypertension. The results obtained indicated that measurement of prothrombin fragment 1+2 could be of use in identifying a "high-risk" group of hypertensive patients who are likely to develop thrombotic events. KEYWORDS: Hypertension, Thrombosis, Hemostasis, Thrombin generation INTRODUCTION Blood pressure is the force of blood against the walls of arteries. Blood pressure rises and falls throughout the day. But when the pressure stays elevated over time, then it is called high blood pressure (hypertension) 1. High blood pressure is dangerous because it makes the heart work too hard and contributes to atherosclerosis (hardening of the arteries). It increases the risk of heart disease and stroke, the first- and third-leading causes of death among Americans. High blood pressure can also results in other conditions, such as congestive heart failure, kidney disease, and blindness 1. Thrombosis often appears to complicate the course of patients with hypertension; thormbosis in some patients with hypertension could be developed to organ damage which makes a dramatic difference to clinical outcome in hypertension 1. Hypertension causes target organ damage by the direct physical effect of increased blood pressure, as well as the active promotion of atherosclerosis and thrombogenesis. More importantly, the processes of thrombogenesis and atherogenesis are intimately related, and many of the basic pathological concepts of thrombogenesis can be applied to atherogenesis 1. Virchow 2 postulated that triads of conditions are needed to predispose to thrombus formation, that is, abnormalities in blood flow, blood constituents, and the vessel wall. Although Virchow 2 was referring to venous thromosis, the same concepts could essentially be applied to arterial 29

2 thrombosis. A modern viewpoint of Virchow s triad includes abnormalities of hemorheology and turbulence at bifurcation and stenotic regions (that is, abnormal blood flow ). Abnormalities in platelets and the coagulation and fibrinolytic pathways ( abnormal blood cons t ituents ) and, finally, abnormalities in the endothelium ( abnor mal vessel wall ). This may explain an important pathophysiological paradox in hypertension, in which despite the blood vessels being exposed to high pressures, the main complications of hypertension are generally thrombotic in nature rather than hemorrhagic 3. Evidence for the prothrombotic or hypercoagulable state in hypertension had been extensively reviewed 4,5. Damage of a target organ makes a dramatic difference to clinical outcome in hypertension. The effects of hypertension are particularly manifested at the heart, brain, kidney, peripheral arteries, and the eye as possible target organs. Hypertensive patients with evidence of target organ damage are well recognized to be at high risk of cardiovascular and cerebrovascular events, and they should be targeted for aggressive blood pressure and risk factor management 5. Indeed, one may postulate that highrisk hypertensive patients with evident damage of target organ are more likely to exhibit a greater prothrombotic or hypercoagulabe state. It is therefore of little surprise that the abnormalities in the prothrombotic or hypercoagulable state in hypertensives have previously been related to the presence of left ventricular hypertrophy on echocardiography 6. 7 A cohort study by Agewall et al found that prothrombin fragment 1+2 levels were independent predictors of major coronary events in treated hypertensive patients. In many previous studies, (Lip, Blann 2000) found that patients with hypertension who developed cardiovascular or cerebrovascular events at 4 years follow-up had a higher baseline von Willebrand factor and fibrin D-dimer levels, although on a Cox multivariate proportional hazards analysis, plasmal fibrinogen and blood pressure levels emerged as independent predictors 8. In their study, Sechi et al 9 staged organ damage in their hypertensives according to WHO guidelines, but it is likely that the abnormalities in various prothrombotic indices can also be related to the degree, and possibly the duration of hypertension. Patients with mild hypertension or lower blood pressures and more recent onset hypertension (which is usually more difficult to precisely quantify) may perhaps show less abnormalities, and such patients are also less likely to have target organ damage. For example, patients with severe hypertension (defined as >160/95 mm Hg) demonstrate high plasma von Willerand factor levels 6, which does not appear to be present in patients with milder elevations of blood pressure 10. the aim of the present study was to determine the prothrombotic or hypercoagulable states among Sudanese hypertensive Patients by measuring thrombin generation markers. MATERIALS and METHODS This is a descriptive, prospective analytical case-control based study conducted in Khartoum State Teaching Hospitals during the period of October 2003 to January 2008 to determine the thrombin generation among Sudanese hypertensive patients. Two hundred patients and fifty normal controls were studied. Patients were those who fulfilled the clinical diagnosis of hypertension of either sex, on or off treatment. The controls were normal, non-hypertensive individuals

3 of either sex. Both patients and control were above 40 years of age. Patients were without previous history of venous or arterial thrombosis, diabetes mellitus. Patients, who received antiplatelet or anticoagulant drugs in the previous 15 days, were excluded from the study. A structured questionnaire consisting of 24 items which was delivered into two parts: Items 1-8 included general information such as, name, residence, occupation, sex, age, duration of the disease, family history, and treatment. Items 9-10 included laboratory investigations that were done to all case-control groups. Platelet poor citrate plasma (PPP) samples were prepared as followed: The blood was drawn using a 20 or 21 G needle with limited occlusion of the arm by the tourniquet. The blood was added to the anticoagulant at a ratio of 4.5 ml of blood to 0.5 ml of citrate (3.2) M buffered sodium citrate) and gently mixed. A bluetopped 3.2 buffered sodium citrate container was used 11. The samples were centrifuged at 1700g for 15 minutes to obtain platelet-poor plasma. The plasma should have a final platelet count that should be less than 10,000 per cubic mm (less than 10x109/L). The plasma was placed into plastic tubes, capped and frozen at-20/-35 C till used for prothrombin fragment (F1.2) and thrombin-antithrombin complex (TAT) determination. TAT and F1+2 levels were determined by enzyme-linked immunosorbent assay, ELISA (Dade -Behring GmbH, Schwalbach, Germany). RESULTS Characteristics of the study population: The age of the patients ranged between 40 to above 69 years with a mean of 58.7 years, the controls had the same age range with the mean 31 of 47.2 years (Fig. (1) and (2) respectively). Eighty nine of the patients(44.5) had stage I hypertension (Systolic or diastolic mm Hg) and 111 of them (55.5) had stage II hypertension (Systolic equal to or more than 160 or diastolic equal to or more than 100 mm Hg). Family History of hypertensive patients: As summarized in table 1 and table 2 there was a significant correlation between hypertension and family history.about 62 of hypertensive patients had a family history of hypertension while only 18 of the control group had family history of the disease. Furthermore, there was no significant correlation between family history of hypertension and the gender of the study group. Duration of hypertension and the treatment: Hypertensive patients in the study group presented with a duration range from 1 month up to 40 years (Fig. 3). About 82.4 of the patients used antihypertensive drugs while 17.6 were on diet and herbal control therapy (Fig. 4). Thrombin generation markers: The marker of thrombin generation, Prothrombin fragment 1+2: mean plasma level of Prothrombin fragment 1+2 measured in hypertensive patients(0.87 nmol/l) was not significantly correlated (p=0.925) to the mean level of normotensive control group (0.88 nmol/l) (table 3). Plasma concentrations of thrombin antithrombin complex, (a marker of the amount of thrombin that was generated). The mean plasma concentration of thrombin antithrombin complex measured in the hypertensive patients (3.04 μg/l) of no significant difference (P= 0.867)

4 compared to the mean level of the control group(2.99 μg/l) Table 4. Table 5 shows that both circulating markers of thrombin generation were significantly(p=0.000) correlated to the duration of hypertension. The age related incidence had increased in Prothrombin fragment 1+2, and thrombin antithrombin complex levels were significantly correlated to the elder patients above 69 years of age Table 6. While prothrombin fragment 1+2 level was significantly (p= 0.012) correlated to the severity of hypertension, thrombin antithrombin complex level did not show significant (p=0.124) correlation to the severity of the disease Table 7. Thrombin antithrombin complex levels between hypertensive patients who, at inclusion, were taking antihypertensive drugs or untreated patients, such treatment did not reveal any difference, (Fig. 5 and 6) Percentage 10 > Age groups Figure 1. Age distribution of hypertensive patients (n, 2003) Percentage 10 > Age groups Figure 2. Age distribution of the control group (n, ) Table 1. Family history of hypertension among patients in the study group (n, 200). Family history Frequency Percentage P.Value Positive Negative Total Table 2. Family history of hypertension in the control group (n, ). Family history Frequency Percentage P.Value Positive 9 18 Negative Total

5 years 6-10 years 1 month- 5 years Percentage of duration Duration of hypertension Figure 3. The duration of hypertension in the study group (n, 200) Percentage of Patients DCH AHD 0 Types of Treatment Figure 4. type of hypertension treatment in the study group (n, 200). AHD: anti-hypertensive drugdch: diet control and herbal therapy Table 3. Mean of prothrombin fragment 1+2 (F1+2) level in the hypertensive patients (n, 200) and the control group (n, ) F 1+2 patient samples No Mean (nmol/l) Std. Deviation P.value control F1+2 : prothrombin fragment 1+2Std: standard Table 4. Mean of thrombin antithrombin complex (TAT) level in the hypertensive patients (n,200) and the control group (n, ) TAT samples No. Mean (μg/l) Std. Deviation P.value patient control TAT: thrombin antithrombin complexstd: standard Table 5. Correlation of the thrombin generation markers and the duration of hypertension in the study group Duration Markers F 1+2(nmol/L) n () 33 TAT (μg/l) n () 1 month-5years 0(0.0) 0(0.0) 6-10 years 0(0.0) 1 (0.5) years 25(15) 33(16.5) Total 25(15) 34(17)

6 P.value Table 6. The age-related incidences to the increased plasma level of the thrombin generation markers in the study group. Age/years Increased plasma level F 1+2(nmol/L) n () TAT(μg/L) n () (0.0) 0(0.0) (0.0) 1(0.5) (3) 3(1.5) > 69 20(12) (15) Total 25(15) 34(17) P.value Table 7. Correlation of the thrombin generation markers and the severity of hypertension in the study group. Hypertension Markers F 1+2(μg/L) n () TAT(μg/L) n () Stage I 1(0.6) 19(9.5) Stage II 24(14.4) 15(7.5) Total 25(15) 34(17) P.value NS NS: non significant Percentage level of F1+2 DCH Types of Treatment AHD 46 Figure 5. Correlation of antihypertensive drugs and untreated patients in F1+2 levels in the study group. DH: anti-hypertensive drugdch: diet control and herbals therapy Percentage level of TAT DCH Types of Treatment AHD 46 Figure 6. Correlation of antihypertensive drugs and untreated patients in TAT levels in the study group. ADH: anti-hypertensive drugdch: diet control and herbals therapy DISCUSSION Prothrombin fragment 1+2 (F1+2) is a polypeptide released from prothrombin during its activation to thrombin by the prothrombinase complex. 34 Measurement of circulating levels of F1+2 was considered a specific marker of thrombin generation Invivo 12,13. Thrombin-antithrombin complexes

7 (TAT) form covalently following thrombin generation. The presence of TAT indicates ongoing thrombin formation and the consumption of antithrombin. The significant association between plasma F1+2 levels and hypertension in the present study indicated a significant activation of the coagulation pathways. In the present study F1+2 was the only hemostatic parameter directly correlated with blood pressure (p=0.012), while TAT was non-significantly correlated with the severity of the disease. The present findings were in agreement with Kienast et.al 4 who suggested that measurement of F1+2 in plasma was more reliable than other markers for ongoing coagulation, such as thrombin-antithrombin complexes. Moreover, the shorter TAT plasma half-life could lead to this possibility. However, whether F1+2 provided a useful marker of atherosclerosis is still debated, which suggests that elevation in PF1+2 might occur as the result of hypertension-induced blood vessel damage. The PF1+2 levels in this study could possibly, indicated systemic thrombin production, providing evidence of in vivo activation of blood coagulation. It was found that plasma F1+2 in the present study in subjects without thrombotic event diseases, proved to be significantly associated with the severity of hypertension. Because F1+2 represents a marker of thrombin generation, the results suggested interrelationships between thrombin generation and progression of arteriosclerosis. This finding may be relevant to a better understanding of the pathophysiological relation between thrombin generation and hypertension. Because thrombin, a central enzyme in the coagulation cascade, is also known to play a role in atherosclerosis progression, 35 measurement of plasma levels of F1+2 might be of value in predicting sub clinical atherosclerosis and in the identification of patients at high vascular risk who may benefit from prophylactic antithrombotic strategies 15,16. The finding of this study tentatively, suggested that interfering with thrombin generation, as assessed by reduction of F1+2, might be a means of reducing the progression of atherosclerotic disease. In conclusion the comparison of treated and untreated hypertensive patients did not show any significant differences in F 1+2 and TAT levels. This finding was in agreement with the results reported by Sechi et.al. 17 who suggested that treatment could be an important confounder in severe hypertension (stage II). There appeared to be little difference in levels of various prothrombotic indices between treated and untreated patients. The half-life of F1+2, approximately 90 minutes, makes its measurement in plasma more reliable than other markers for ongoing coagulation, such as thrombin-antithrombin complexes which are considered as hypertensioninduced blood vessel damage. ACKNOWLEDGMENT One of us, fathelrahman M.H, I would like to thank Prof. Michel C. Perry for giving him the opportunity to work with his group at Ellis Fischer Medical Center, Columbia, Missouri, USA. His group contributed essentially to the final outcome of the practical studies, prof. perry insights and comments were invaluable over the time. The euothers are indebted to the group at the Missouri Medical Center especially Dr. Kathy Olson and Dr. Raymond Lobins since this research project benefited a lot from their critical discussions.

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