**4.3 Validation of the VCH thesis**

The original VCH hypothesis led to two readily testable predictions, which were the subjects of experimental studies during the 1980s. The results of these studies were unequivocal:


The first prediction was examined on a series of patients undergoing surgery for varicose veins (Hamer *et al.*, 1981). In accordance with standard practice, the anaesthetist maintained normal circulating PO2 levels in these patients. However, the PO2 in the valve pockets fell consistently, and when the oxygen electrode was brought into contact with the endothelial surface lining at the base of the pockets, there was no response. The PO2 level in these cells had fallen to a value below the detection limit of the electrode, not only confirming the predicted hypoxaemia in the valve pocket blood during sustained non-pulsatile flow (the circulating blood being still normally oxygenated), but *a fortiori* showing that the valve cusp leaflet is effectively impermeable to oxygen.

The second prediction was tested on dogs that breathed normally after a single dose anaesthetic injection, which by paralysing all muscle-pumping other than respiratory excursions ensured that blood flow in the limbs was non-pulsatile (Hamer & Malone, 1984). When the dogs began to awaken at successive intervals, the legs exhibited 'auto-jactitation' (clonus), each spasm sufficient to pump fresh blood containing living leukocytes and platelets into the recently hypoxaemic valve pockets. X-rays of the upper thigh veins using contrast radiography then revealed the growth of thrombi, which were microscopically/ morphologically indistinguishable from autochthonous venous thrombi.

The significance of these experimental findings was acknowledged by Hume (1985), who stated that the aetiology of DVT was now elucidated. The explanation of venous thrombogenesis in terms of valve cusp hypoxia was empirically demonstrated, no longer a mere hypothesis. (Hume, a surgeon who specialised in DVT, had co-authored a major monograph on the subject with Sevitt and Thomas in 1970.)

There is a third prediction, which without the VCH thesis might seem counterintuitive: it is that thrombi may form in limb veins should *rapid* blood flow remain non-pulsatile for extended periods. This prediction could repay testing on experimental animals; a convincing experiment would be instructive, though it would be technically difficult to conduct.

Pathophysiology and Clinical Aspects of 142 Venous Thromboembolism in Neonates, Renal Disease and Cancer Patients

serial deposition of white cells may contribute the most distinctive morphological

Subsequent dehiscence of the growing thrombus, with or without the necrotic endothelial layer to which it is attached, might explain why venous thrombi embolise so readily. The VCH thesis also provides an aetiological explanation for post-thrombotic syndrome and,

The original VCH hypothesis led to two readily testable predictions, which were the subjects of experimental studies during the 1980s. The results of these studies were unequivocal:

1. deliberately (artificially) sustained non-pulsatile flow in human patients under anaesthesia causes extreme hypoxaemia in the valve pockets, which results in hypoxia extending to anoxia in the blood 'servicing' endothelial cells in the distal pockets; 2. sustained non-pulsatile flow alternating with brief episodes of pulsatile flow in experimental animals, also under anaesthesia, generates thrombi that are

The first prediction was examined on a series of patients undergoing surgery for varicose veins (Hamer *et al.*, 1981). In accordance with standard practice, the anaesthetist maintained normal circulating PO2 levels in these patients. However, the PO2 in the valve pockets fell consistently, and when the oxygen electrode was brought into contact with the endothelial surface lining at the base of the pockets, there was no response. The PO2 level in these cells had fallen to a value below the detection limit of the electrode, not only confirming the predicted hypoxaemia in the valve pocket blood during sustained non-pulsatile flow (the circulating blood being still normally oxygenated), but *a fortiori* showing that the valve cusp

The second prediction was tested on dogs that breathed normally after a single dose anaesthetic injection, which by paralysing all muscle-pumping other than respiratory excursions ensured that blood flow in the limbs was non-pulsatile (Hamer & Malone, 1984). When the dogs began to awaken at successive intervals, the legs exhibited 'auto-jactitation' (clonus), each spasm sufficient to pump fresh blood containing living leukocytes and platelets into the recently hypoxaemic valve pockets. X-rays of the upper thigh veins using contrast radiography then revealed the growth of thrombi, which were microscopically/

The significance of these experimental findings was acknowledged by Hume (1985), who stated that the aetiology of DVT was now elucidated. The explanation of venous thrombogenesis in terms of valve cusp hypoxia was empirically demonstrated, no longer a mere hypothesis. (Hume, a surgeon who specialised in DVT, had co-authored a major

There is a third prediction, which without the VCH thesis might seem counterintuitive: it is that thrombi may form in limb veins should *rapid* blood flow remain non-pulsatile for extended periods. This prediction could repay testing on experimental animals; a convincing

experiment would be instructive, though it would be technically difficult to conduct.

morphologically indistinguishable from autochthonous venous thrombi.

monograph on the subject with Sevitt and Thomas in 1970.)

morphologically indistinguishable from autochthonous venous thrombi.

indeed, for chronic venous insufficiency in general (Malone & Agutter, 2009).

characteristic of a venous thrombus, the striking Lines of Zahn.

**4.3 Validation of the VCH thesis** 

leaflet is effectively impermeable to oxygen.

Fig. 1. Blood movements within and around a venous valve pocket during normal (pulsatile flow) conditions. The blood in the pocket exchanges regularly with the luminal blood, so valve pocket hypoxaemia does not develop and the endothelia lining the pocket remain oxygenated.

Aetiology of Deep Venous Thrombosis - Implications for Prophylaxis 145

Fig. 3. Each single pulsation by the muscle pump will evacuate 'dead blood' from all affected valve pockets after prolonged periods of hypoxaemia and replace it with fresh blood containing living, active white cells including platelets. In this diagrammatic

& van der Vleet (2011), who propose that coagulation is initiated on the vein wall

section 5.

endothelium, not the valve cusp parietalis, a speculation that will be discussed further in

illustration, these fresh cells are shown in juxtaposition with the parietalis endothelium; our evidence and the historical literature (Aschoff, 1924; Saphir & Lev, 1952a; Sevitt, 1974) indicate that leukocytes swarm on to the severely embarrassed or possibly dead/ necrotic cell layer, forming the white core of the thrombus *Kopfteil*. Because the valve leaflet projects into the centre of the vein, the *Kopfteil* may have seemed to its first observers to be 'in the centre of the afflicted vein'. It may be conjectured that the vein wall endothelial cells deep in the valve pocket also become sufficiently hypoxic for the egr-1 pathway to be activated, notwithstanding any possible oxygenation via the vasa venarum. This is assumed by Bovill

Fig. 2. When blood flow along the vein becomes non-pulsatile, irrespective of speed, the blood in the valve pocket is no longer exchanged with luminal blood, as it is under normal pulsatile flow conditions (Fig. 1). The laminar flow past the mouth of the valve pocket drives a vortex (A) in the upper part of the pocket, and this in turn drives a secondary vortex (B), rotating very slowly in the opposite sense, in the depths of the pocket. Local hypoxaemia leads to severe hypoxia, especially of the parietalis endothelium.

Pathophysiology and Clinical Aspects of 144 Venous Thromboembolism in Neonates, Renal Disease and Cancer Patients

Fig. 2. When blood flow along the vein becomes non-pulsatile, irrespective of speed, the blood in the valve pocket is no longer exchanged with luminal blood, as it is under normal pulsatile flow conditions (Fig. 1). The laminar flow past the mouth of the valve pocket drives a vortex (A) in the upper part of the pocket, and this in turn drives a secondary vortex (B), rotating very slowly in the opposite sense, in the depths of the pocket. Local

hypoxaemia leads to severe hypoxia, especially of the parietalis endothelium.

Fig. 3. Each single pulsation by the muscle pump will evacuate 'dead blood' from all affected valve pockets after prolonged periods of hypoxaemia and replace it with fresh blood containing living, active white cells including platelets. In this diagrammatic illustration, these fresh cells are shown in juxtaposition with the parietalis endothelium; our evidence and the historical literature (Aschoff, 1924; Saphir & Lev, 1952a; Sevitt, 1974) indicate that leukocytes swarm on to the severely embarrassed or possibly dead/ necrotic cell layer, forming the white core of the thrombus *Kopfteil*. Because the valve leaflet projects into the centre of the vein, the *Kopfteil* may have seemed to its first observers to be 'in the centre of the afflicted vein'. It may be conjectured that the vein wall endothelial cells deep in the valve pocket also become sufficiently hypoxic for the egr-1 pathway to be activated, notwithstanding any possible oxygenation via the vasa venarum. This is assumed by Bovill & van der Vleet (2011), who propose that coagulation is initiated on the vein wall endothelium, not the valve cusp parietalis, a speculation that will be discussed further in section 5.

Aetiology of Deep Venous Thrombosis - Implications for Prophylaxis 147

the effect of ageing (cf. Saphir & Lev, 1952b; Van Langevelde *et al*., 2010), and it is consistent with the readiness of venous thrombi to embolise. However, though it is consistent with them, some might reserve judgment as to whether it accounts fully and explicitly for (1) the high density of the fibrin around the *Kopfteil* and (2) the Lines of Zahn. These observations

The dense fibrin in nascent thrombi was likewise described by Sevitt (1974). Bovill & van der Vliet (2011) pointed out that it is consistent with tissue factor (TF)-induced coagulation, but they suggested that coagulation is initiated on the vein wall not the parietalis endothelium of the valve pocket. That suggestion would have to be experimentally verified and corroborated before it could be regarded as fact rather than conjecture. Its plausibility depends on the commonly held belief that TF is the primary physiological trigger for coagulation (e.g. Hoffman & Monroe, 2001); also, TF is one of the many targets of activation by egr-1 (Mechtcheriakova *et al.*, 1999) so it is expected that valve pocket hypoxaemia will activate it. Primary involvement of TF in coagulation during venous thrombogenesis could explain why

anticoagulants are allegedly more effective for prophylaxis than are platelet inhibitors.

blood, and densely crowded platelets will generate a dense fibrin mesh.

weaving the leukocyte/platelet-rich and dense fibrin layers around each other.

**5.2 Cross-talk between leukocyte recruitment and coagulation** 

A possible difficulty with this explanation for the high fibrin density is that heparin does not inhibit the initiation of thrombi on hypoxic venous endothelium (Samuels & Webster, 1952), and heparin is known to inhibit TF directly and by activating tissue factor promoting inhibitor (e.g. Lupu *et al*., 1999; Ettalaie *et al*., 2011). Alternative explanations should therefore be considered. For instance, the inception and growth of a venous thrombus are slow processes, characterised by the serial margination of successive layers of platelets and leukocytes on the hypoxia-induced lesion of the valve pocket endothelium as blood continues to circulate past the site. This results in a much denser crowding of platelets (as well as leukocytes) than is likely in a haemostatic plug or during the coagulation of shed

No matter whether coagulation is TF-induced by the luminal endothelial cells of the valve pocket, as Bovill & van der Vliet speculate, or whether the crowded platelets on and around the injured/ necrotic parietalis endothelium spin out the dense fibrin, logical extension of the VCH thesis provides an explanation for that facet of Virchow's *real* triad. As for the Lines of Zahn, the serial deposition described earlier is the critical factor; but the slow secondary vortex in the depths of the valve pocket (Karino, 1986) will also contribute by

Thus, the extended VCH mechanism accounts for all three aspects of Virchow's *real* triad.

The molecular changes in endothelial cells subjected to valve pocket hypoxaemia were discussed in chapter 12 of Malone & Agutter (2008) and by Bovill & van der Vliet (2011). The generation of reactive oxygen species (ROS) in the hypoxic endothelium is of particular interest because ROS promote both neutrophil recruitment (Millar *et al*., 2007) and the activation of TF (Banfi *et al*., 2009), as well as activating egr-1. This involvement of ROS could support the proposal that a vegan diet has prophylactic value against DVT and VTE (Cundiff *et al.*, 2010), since vegan diets are allegedly rich in antioxidants, though a statistically sound test of this proposal would involve a very large patient cohort. Bovill & van der Vliet note that other hypoxia-related transcription factors such as hypoxia-inducible

need further clarification.

Fig. 4. When sustained non-pulsatile flow resumes after a pulsatile episode, any white cells that have swarmed over the necrotic parietalis endothelium die likewise as a result of valve pocket hypoxaemia. Meanwhile, coagulation can continue. With further episodes of pulsatile flow, the process will be repeated, generating successive layerings of dead cells interspersed with fibrin, accounting for the Lines of Zahn morphology. The necrotic parietalis is fragile and is liable to dehisce (see illustrations in Malone & Agutter, 2008, chapter 10), particularly when the nascent thrombus outgrows the valve pocket and protrudes into the vein lumen, where it is subjected to tension by the flowing blood after the resumption of pulsatility. This may explain the tendency of venous thrombi to embolise.
