**3.2.3 Why do leukocytes accumulate at sites of venous thrombogenesis?**

Hunter realised in 1793, more than 60 years before the germ theory of disease was articulated, that leukocytes swarm to sites of either tissue injury or local infection (a phenomenon more recently termed 'margination of leukocytes'). After Pasteur's work had been accepted, it was widely supposed that infections contribute to the causation of DVT; but when no consistent correlation between thrombosis and infectious agents was established, and antibiotics did not alleviate or prevent DVT, that hypothesis was abandoned during the first half of the 20th century. This aspect of history was reviewed briefly in our monograph (Malone & Agutter, 2008, chapter 7). Once again, Virchow was uncannily prescient on this topic; although unaware of the epoch-making discoveries being made in Pasteur's laboratory while he was writing *Thrombose und Embolie*, he perceptively described the accumulation of white material in thrombi as '*puriform but not purulent'* (Virchow, 1856).

For more than a century there has been experimental support for the inference that the cause of leukocyte and platelet margination at the site of venous thrombogenesis is local injury. Welch (1887, 1899) showed that experimental thrombi induced by electrical or other traumatic injury to the venous endothelium morphologically resembled autochthonous thrombi. This established the only valid way of evaluating experimental thrombi; as stated in section 3.2.1, coagula that lack the structure summarised in Virchow's *real* triad are not 'thrombi'.

The studies of Sandison (1931) and Stewart *et al*. (1974) supported Welch's conclusion: vessel injury (by whatever agency) causes the generation of thrombi indistinguishable from autochthonous ones - dominated by the accumulation of vast numbers of sequestrated/marginated white cells.

However, in most DVT cases encountered clinically and viewed microscopically, the venous endothelium appears ostensibly intact. Therefore, the key questions arising from the studies of Virchow and others (notably Welch and Aschoff) are: what causes the putative *subtle* injury to the venous endothelium that could initiate autochthonous thrombogenesis, and is a particular zone or area of venous endothelium involved?

#### **3.2.4 A caveat**

These two questions were the points of departure for the VCH hypothesis and their answers are fundamental to the VCH mechanism. However, Virchow's *real* triad does not only concern leukocyte/platelet margination at the site of thrombus formation. Its other

Aetiology of Deep Venous Thrombosis - Implications for Prophylaxis 139

micrographs showed thrombi seated in the valve pockets. In some, the exact site of attachment to the endothelium is not clear because the entire pocket is filled with the thrombus, but in several images the thrombus is evidently attached to the inner (parietalis) face of the valve cusp leaflet, not to the outer (luminalis) face, and not to the vein wall. This recurrent observation played a crucial part in the formulation of the VCH

Bovill & van der Vliet (2011), examining the small sample of micrographs in Sevitt (1974), inferred instead that the thrombi were attached to the vein wall endothelium within the valve pocket. We shall discuss this divergence of opinion later; but it seems improbable that a thrombus perceived as 'anchored' in mid-lumen could be attached to the vein wall

The foregoing background led us to articulate the VCH hypothesis and subsequently to

In those mid-20th century papers, it was debated whether oxygen was supplied to the venous endothelium from the luminal blood or the vasa venarum. Presumably, hypoxia sufficiently severe to kill the endothelial cells and induce leukocyte swarming and phagocytosis of the debris required impaired oxygenation from both sources; it is well established that the venous endothelium can survive moderately prolonged hypoxia (Jackson *et al.*, 1988), depending on anaerobic glycolysis to provide ATP (Berna *et al.*, 2001), and surgeons procure the use of a bloodless field under a 2.5 hour tourniquet. However, hypoxia that is not so severe as to kill the cells but is sufficient to alter their phenotype can also lead to leukocyte and platelet recruitment and local coagulation. During the period 1980-2000, studies in a number of laboratories established that significant but non-fatal hypoxia in cultured venous endothelial cells induces the expression of the early growth response-1 (egr-1) gene, and this unleashes a cascade of gene-expression and phenotypic changes that would promote local coagulation and leukocyte accumulation *in vivo* (Pinsky *et* 

*al.*, 1995; Yan *et al.*, 1999a,b; Karimova & Pinsky, 2001; Ten & Pinsky, 2002).

**4.1 What severely embarrasses or kills the endothelium in a venous valve pocket?**  The clue to what injures the endothelial lining of an intact, functioning, vein was suggested by Drinker (1938) and van Ottingen (1941), whose researches unexpectedly established that copious and ubiquitous thrombus-like coagula formed in the veins of the victims of carbon monoxide poisoning and were associated with endothelial alterations. Evidently, either carboxyhaemoglobinaemia or hypoxaemia could cause the endothelial injury associated with DVT. Drinker proposed this hypothesis to O'Neill (1947) who provisionally validated it, and Samuels & Webster (1952) supported the proposal. Malone & Morris (1978) showed that lesions similar to the white parts of autochthonous and experimental thrombi formed in the veins of oxygen-starved animals by the process of margination and sequestration of platelets and leukocytes. Interestingly, Samuels & Webster (1952) showed that heparin does not prevent the development of a nascent

**4. Valve Cusp Hypoxia: The basis of venous thrombogenesis** 

thrombus on a hypoxically injured endothelium.

hypothesis.

endothelium.

validate it.

two microscopic facets should not be overlooked: the remarkably high density of fibrin in those parts of a thrombus that are formed first, in the *Kopfteil*; and the extraordinary morphology of the Lines of Zahn. These observations remind us that despite the importance of leukocytes in thrombogenesis, their swarming around the site coincides with, and is often preceded by, local coagulation. In the tenth lecture in *Die Cellularpathologie*, Virchow (1858) overtly attacked Cruveilhier but made this exception: *"Cruveilhier was right... that the so-called pus in the veins never, in the first instance, lies against the wall of the vein, but is always seen first in the centre of a previously existing coagulum which marks the start of the process".* 

This observation indicates that the subtle endothelial injury inducing local leukocyte/platelet swarming and margination also initiates local coagulation, which may proceed rapidly. But though Virchow did not speculate about the cause or nature of that subtle injury, he did pin-point its location.
