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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Physiol Funct Imaging. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2839247
NIHMSID: NIHMS183502

Early activation of the coagulation system during lower body negative pressure

Summary

We considered that a moderate reduction of the central blood volume (CBV) may activate the coagulation system. Lower body negative pressure (LBNP) is a non-invasive means of reducing CBV and, thereby, simulates haemorrhage. We tested the hypothesis that coagulation markers would increase following moderate hypovolemia by exposing 10 healthy male volunteers to 10 min of 30 mmHg LBNP. Thoracic electrical impedance increased during LBNP (by 2·6 ± 0·7 Ω, mean ± SD; P < 0·001), signifying a reduced CBV. Heart rate was unchanged during LBNP, while mean arterial pressure decreased (84 ± 5 to 80 ± 6 mmHg; P < 0·001) along with stroke volume (114 ± 22 to 96 ± 19 ml min−1; P < 0·001) and cardiac output (6·4 ± 2·0 to 5·5 ± 1·7 l min−1; P < 0·01). Plasma thrombin–antithrombin III complexes increased (TAT, 5 ± 6 to 19 ± 20 μg l−1; P < 0·05), indicating that LBNP activated the thrombin generating part of the coagulation system, while plasma D-dimer was unchanged, signifying that the increased thrombin generation did not cause further intravascular clot formation. The plasma pancreatic polypeptide level decreased (13 ± 11 to 6 ± 8 pmol l−1; P < 0·05), reflecting reduced vagal activity. In conclusion, thrombin generation was activated by a modest decrease in CBV by LBNP in healthy humans independent of the vagal activity.

Keywords: haemorrhage, hypovolemia, pancreatic polypeptide, thrombin generation, vagal activity

Introduction

We considered that the coagulation system is activated by a reduction of the central blood volume (CBV). Both patients with a ruptured abdominal aortic aneurysm (Skagius et al., 2008) and trauma demonstrate a procoagulant state that could render the patients better protected against continued haemorrhage (Innes & Sevitt, 1964; Kaufmann et al., 1997). It is, however, unknown whether even moderate hypovolemia affects the coagulation system by an increase in either parasympathetic or sympathetic activity (Kuznik & Mishchenko, 1975a; von Känel & Dimsdale, 2000).

Lower body negative pressure (LBNP) pools blood in the abdomen and lower extremities thereby reducing the CBV (Murray et al., 1968) as indicated by an increase in thoracic electrical impedance and reduced leg impedance (Cai et al., 2000; Kitano et al., 2005). Hence, LBNP is a non-invasive human model for central hypovolemia (Cooke et al., 2004) and provides a means to assess the effect of a reduced CBV on coagulation markers. We tested the hypothesis that a moderate decrease of CBV during LBNP activates the coagulation system in healthy humans.

Methods

The study was conducted in accordance with the Helsinki declaration and approved by the Ethical Comity of Copenhagen and Frederiksberg (KF 11 312841) and 10 healthy male volunteers (26 ± 3 years, 80 ± 11 kg and 182 ± 6 cm) provided informed consent prior to commencement of study related activities. The subjects reported to the laboratory at 9 AM after an overnight fast. A cannula (1·1 mm ID, 20 gauge) was placed in the brachial artery of the non-dominant arm for blood sampling and measurement of mean arterial pressure (MAP) with a Bentley transducer (Uden, The Netherlands) positioned at the level of the midaxillary line and connected to a Dialogue 2000 monitor (IBC-Danica, Copenhagen, Denmark). A three-lead electrocardiogram recorded heart rate (HR), while stroke volume (SV) and cardiac output (CO) were estimated with a Finometer (Finapres Medical System BV, Amsterdam, The Netherlands). Such assessment of CO has been successfully validated both in healthy subjects and in patients during surgery and sepsis (Jellema et al., 1999; Bogert & Van Lieshout, 2005; Nissen et al., 2009). Cardiovascular variables were obtained as averaged data from 10–15 min before LBNP, at 5 and 10 min of LBNP, and ~5 min thereafter. Thoracic electrical impedance indicates changes in CBV; i.e. when the blood volume decreases, electrical impedance increases (Cai et al., 2000). Paired electrodes were placed on the right sternocleidomastoid muscle and on the upper left rib at the mid-axillary line and evaluation was based upon a 200 μA current at 1·5 and 100 kHz (C-Guard, Danmeter, Denmark).

After instrumentation, the subject was placed supine in the LBNP chamber sealed at the level of the iliac crest. To reduce the risk of lower-back pain and stiffness and to reduce body movements, a wedge shaped pillow was placed under the knees. Following 30 min of supine rest, the subject was exposed to 10 min of 30 mmHg LBNP. Arterial blood (~3 ml) was obtained 10–15 min before LBNP and at 5 and 10 min of LBNP, and sampled into citrate tubes.

Blood samples were centrifuged at 1000 g for 10 min at 20°C within 30 min of sampling and plasma was stored at −80°C for less than 3 months. Plasma was analysed for markers of hemostatic procoagulant activity: Thrombin–antithrombin III complexes (TAT; Enzygnost®; Dade Behring, Marburg, Germany) as a measure of thrombin generation; D-dimer (Tina-quant, cobas®; Roche Diagnostics GmbH, Mannheim, Germany) that is a fibrinolytic product; and for prothrombin time (PT; Nycotest®; PT, Axis-Shield, Oslo, Norway) evaluating the tissue factor dependent pathway of the coagulation system, and by activated partial thromboplastin time (aPTT; Platelin®; LS, bioMèrieux, Inc., NC, USA) to evaluate the platelet dependent pathway (Futura, Instrumentation Laboratory, Milan, Italy). Plasma pancreatic polypeptide was determined by radioimmunoassay with a polyclonal rabbit-antibody to indicate vagal activity (Schwartz et al., 1978; Damholt et al., 1997).

Data analysis

Cardiovascular variables were obtained with a Biopac acquisition system (MP100 Acknowledge, Biopac Systems Inc., CA, USA). Data were evaluated by one-way repeated measures ANOVA followed by a Student–Newman–Keuls post hoc test using SigmaStat (version 3.0). If the normal distribution test failed, a Friedman repeated measures ANOVA on Ranks was applied. A P-value <0·05 was considered to represent a statistical significant difference and data are presented as mean ± SD.

Results

Thoracic electrical impedance increased during LBNP (by 2·6 ± 0·7 Ω; P < 0·001). While HR was unaffected at 55 ± 7 bpm, there was a decrease in MAP (84 ± 5 to 80 ± 6 mmHg; P < 0·001) along with SV (114 ± 22 to 92 ± 21 ml min−1; P < 0·001) and therefore CO (6·4 ± 2·0 to 5·5 ± 1·7 l min−1; P < 0·01). Thoracic electrical impedance was still elevated 5 min after termination of LBNP (0·5 ± 0·3 Ω; P < 0·01), but the cardiovascular variables had returned to baseline levels (84 ± 5 mmHg, 115 ± 25 ml min−1; and 6·6 ± 2·3 l min−1). While plasma pancreatic polypeptide decreased after 10 min of LBNP (13 ± 11 to 6 ± 8 pmol l−1; P < 0·05), plasma TAT complexes increased ~5-fold (5 ± 6 to 27 ± 36 μg l−1; P < 0·05) after 5 min of LBNP but decreased slightly hereafter (to 19 ± 20 μg l−1; P < 0·05) (Fig. 1). Plasma aPTT decreased marginally after 10 min of LBNP (31·3 ± 3·2 to 30·7 ± 3·1 s; P < 0·05), while plasma PT and D-dimer were unchanged (0·80 ± 0·10 U ml−1 and 0·10 ± 0·08 μg ml−1 respectively).

Figure 1
Thrombin–antithrombin III (TAT) complexes, D-dimer, activated partial thromboplastin time (aPTT), and prothrombin time (PT) for 10 healthy male volunteers before and following 5 and 10 min of 30 mmHg lower body negative pressure (LBNP). Values ...

Discussion

Haemorrhage following injury is a major cause of death and establishing hemostasis is an important survival strategy. The coagulation system is activated following injury (Meissner et al., 2003) and the vasovagal syncope may be taken to represent a last line of defence against bleeding (Diehl, 2005), because vagal activity provokes activation of the coagulation system (Kuznik & Mishchenko, 1975a,b). Indeed, some trauma patients in hemorrhagic shock demonstrate parasympathetic activation as indicated by bradycardia and increased plasma pancreatic polypeptide (Sander-Jensen et al., 1986b). We evaluated the displacement of blood to the lower extremities by thoracic electrical impedance and estimated that 0·3–1·0 l of fluid was displaced from the chest to the splanchnic region and the legs (Murray et al., 1968; Cooke et al., 2004).

LBNP reduced SV and CO by 20% and 15% respectively, and also MAP, but HR was unaffected. Plasma pancreatic polypeptide decreased indicating that vagal activity was reduced in accordance with the initial response to head-up tilt (Shi et al., 2000). Nevertheless, upon exposure to LBNP, plasma TAT complexes increased to a level similar to that reported in patients with deep vein thrombosis or suspected disseminated intravascular coagulation (Hoek et al., 1988), indicating elevated thrombin generation. Thrombin is a component in clot formation and, therefore, indicates the activity of the coagulation system (Roberts et al., 2006). Notably, the plasma D-dimer concentration did not change indicating that the elevated thrombin generation did not provoke stable clot formation. D-dimer is a degradation product of crosslinked fibrin, i.e. a stable clot and is applied for the diagnosis of deep-vein thrombosis and venous thromboembolism (Bounameaux et al., 1994). Also, PT was unaffected and aPTT decreased slightly suggesting that increased thrombin formation did not result in a significant consumption of coagulation factors.

Plasma pancreatic polypeptide indicated that vagal activity decreased during LBNP, thus parasympathetic activity cannot explain the increase in plasma TAT complexes. The sympathetic nervous system may also enhance coagulation, e.g. adrenaline enhances hemostatic activity (von Känel & Dimsdale, 2000), and the plasma adrenaline level may increase 3-fold during LBNP and head-up tilt (Sander-Jensen et al., 1986a, 1988). While it is unlikely that adrenaline was increased to such a level in our test subjects, the applied level of LBNP is sufficient to increase muscle sympathetic nerve activity (Rea & Wallin, 1989).

The blood pooling in the legs during LBNP also resembles the pooling that happens during prolonged upright immobility, e.g. prolonged sitting during air and bus travel, where 0·2–0·5 l of blood may pool in the lower extremities (Mittermayr et al., 2003; Schobersberger et al., 2004). During such long-distance travel, the coagulation system is activated and there is an increase in venous thromboembolism (Homans, 1954; Schobersberger et al., 2004). While the activation of the coagulation system is shown after hours of travel, only 15 min of still standing activates the coagulation (Masoud et al., 2008), which is in accordance with the activation of the coagulation after 5–10 min of LBNP in the present study. During pooling of the blood in the lower extremities, the coagulation may be activated because of increased orthostatic stress on the endothelial monolayer in the lower extremities that stimulates the expression of tissue factor, release of von Willebrand factor, and increases the levels of tissue plasminogen activator and plasminogen activator inhibitor-1 (Masoud et al., 2008).

In summary, LBNP-induced reduction in CBV, similar to mild to moderate haemorrhage, activates the coagulation system as indicated by increased plasma TAT complex formation unrelated to parasympathetic activity. Thus, like the circulatory system, the coagulation system responds early to a reduced CBV.

References

  • Bogert LW, Van Lieshout JJ. Non-invasive pulsatile arterial pressure and stroke volume changes from the human finger. Exp Physiol. 2005;90:437–446. [PubMed]
  • Bounameaux H, De Moerloose P, Perrier A, Reber G. Plasma measurement of D-dimer as diagnostic aid in suspected venous thromboembolism: an overview. Thromb Haemost. 1994;71:1–6. [PubMed]
  • Cai Y, Boesen M, Strømstad M, Secher NH. An electrical admittance based index of thoracic intracellular water during head-up tilt in humans. Eur J Appl Physiol. 2000;83:356–362. [PubMed]
  • Cooke WH, Ryan KL, Convertino VA. Lower body negative pressure as a model to study progression to acute hemorrhagic shock in humans. J Appl Physiol. 2004;96:1249–1261. [PubMed]
  • Damholt M, Rasmussen BK, Hilsted L, Jensen R, Hilsted J. Basal serum pancreatic polypeptide is dependent on age and gender in an adult population. Scand J Clin Lab Invest. 1997;57:695–702. [PubMed]
  • Diehl RR. Vasovagal syncope and Darwinian fitness. Clin Auton Res. 2005;15:126–129. [PubMed]
  • Hoek JA, Sturk A, ten Cate JW, Lamping RJ, Berends F, Borm JJ. Laboratory and clinical evaluation of an assay of thrombin–antithrombin III complexes in plasma. Clin Chem. 1988;34:2058–2062. [PubMed]
  • Homans J. Thrombosis of the deep leg veins due to prolonged sitting. N Engl J Med. 1954;250:148–149. [PubMed]
  • Innes D, Sevitt S. Coagulation and fibrinolysis in injured patients. J Clin Pathol. 1964;17:1–13. [PMC free article] [PubMed]
  • Jellema WT, Wesseling KH, Groeneveld AB, Stoutenbeek CP, Thijs LG, Van Lieshout JJ. Continuous cardiac output in septic shock by simulating a model of the aortic input impedance: a comparison with bolus injection thermodilution. Anesthesiology. 1999;90:1317–1328. [PubMed]
  • von Känel R, Dimsdale JE. Effects of sympathetic activation by adrenergic infusions on hemostasis in vivo. Eur J Haematol. 2000;65:357–369. [PubMed]
  • Kaufmann CR, Dwyer KM, Crews JD, Dols SJ, Trask AL. Usefulness of thrombelastography in assessment of trauma patient coagulation. J Trauma. 1997;42:716–720. [PubMed]
  • Kitano A, Shoemaker JK, Ichinose M, Wada H, Nishiyasu T. Comparison of cardiovascular responses between lower body negative pressure and head-up tilt. J Appl Physiol. 2005;98:2081–2086. [PubMed]
  • Kuznik BI, Mishchenko VP. Effect of vagus nerve stimulation on coagulability of the blood in cats. Bull Exp Biol Med. 1975a;77:723–725. [PubMed]
  • Kuznik BI, Mishchenko VP. The role of the vascular wall in the mechanism of control of blood coagulation and fibrinolysis on stimulation of the vagus nerve. Cor Vasa. 1975b;17:151–158. [PubMed]
  • Masoud M, Sarig G, Brenner B, Jacob G. Orthostatic hypercoagulability: a novel physiological mechanism to activate the coagulation system. Hypertension. 2008;51:1545–1551. [PubMed]
  • Meissner MH, Chandler WL, Elliott JS. Venous thromboembolism in trauma: a local manifestation of systemic hypercoagulability? J Trauma. 2003;54:224–231. [PubMed]
  • Mittermayr M, Fries D, Innerhofer P, Schobersberger B, Klingler A, Partsch H, Fischbach U, Gunga HC, Koralewski E, Kirsch K, Schobersberger W. Formation of edema and fluid shifts during a long-haul flight. J Travel Med. 2003;10:334–339. [PubMed]
  • Murray RH, Thompson LJ, Bowers JA, Albright CD. Hemodynamic effects of graded hypovolemia and vasodepressor syncope induced by lower body negative pressure. Am Heart J. 1968;76:799–811. [PubMed]
  • Nissen P, Van Lieshout JJ, Novovic S, Bundgaard-Nielsen M, Secher NH. Techniques of cardiac output measurement during liver transplantation: arterial pulse wave versus thermodilution. Liver Transpl. 2009;15:287–291. [PubMed]
  • Rea RF, Wallin BG. Sympathetic nerve activity in arm and leg muscles during lower body negative pressure in humans. J Appl Physiol. 1989;66:2778–2781. [PubMed]
  • Roberts HR, Hoffman M, Monroe DM. A cell-based model of thrombin generation. Semin Thromb Hemost. 2006;32:32–38. [PubMed]
  • Sander-Jensen K, Secher NH, Astrup A, Christensen NJ, Giese J, Schwartz TW, Warberg J, Bie P. Hypotension induced by passive head-up tilt: endocrine and circulatory mechanisms. Am J Physiol Regul Integr Comp Physiol. 1986a;251:R742–R748. [PubMed]
  • Sander-Jensen K, Secher NH, Bie P, Warberg J, Schwartz TW. Vagal slowing of the heart during haemorrhage: observations from 20 consecutive hypotensive patients. Br Med J. 1986b;292:364–366. [PMC free article] [PubMed]
  • Sander-Jensen K, Mehlsen J, Stadeager C, Christensen NJ, Fahrenkrug J, Schwartz TW, Warberg J, Bie P. Increase in vagal activity during hypotensive lower-body negative pressure in humans. Am J Physiol Regul Integr Comp Physiol. 1988;255:R149–R156. [PubMed]
  • Schobersberger W, Mittermayr M, Innerhofer P, Sumann G, Schobersberger B, Klingler A, Simmer M, Streif W, Fischbach U, Fries D. Coagulation changes and edema formation during long-distance bus travel. Blood Coagul Fibrinolysis. 2004;15:419–425. [PubMed]
  • Schwartz TW, Holst JJ, Fahrenkrug J, Jensen SL, Nielsen OV, Rehfeld JF, de Muckadell OB, Stadil F. Vagal, cholinergic regulation of pancreatic polypeptide secretion. J Clin Invest. 1978;61:781–789. [PMC free article] [PubMed]
  • Shi X, Wray DW, Formes KJ, Wang HW, Hayes PM, Yurvati AH, Weiss MS, Reese IP. Orthostatic hypotension in aging humans. Am J Physiol Heart Circ Physiol. 2000;279:H1548–H1554. [PubMed]
  • Skagius E, Siegbahn A, Bergqvist D, Henriksson A. Activated coagulation in patients with shock due to ruptured abdominal aortic aneurysm. Eur J Vasc Endovasc Surg. 2008;35:37–40. [PubMed]