**3.1.3 Microparticles, VTE and cancer**

In contrast to the above discussion for idiopathic VTE – thrombosis, cancer and microparticles seem to have a more definitive relationship. The MP are thought to reflect a balance between cell stimulation, proliferation and death which may be important in cancer related thrombosis. Cancer increases the risk of VTE by four fold and addition of chemotherapy further increases the risk by six to eight fold (Furie and Furie 2006). It is possible that circulating MP shed from cancer cells represent an indication for tumours to metastasize in the absence of any other clinical evidence for metastasis. A recent report states that platelet MP markedly stimulated the metastatic potential of 5 different cancer cell lines (Rak). It has also been shown that human tumor derived MP when injected into mice activated coagulation by virtue of their TF procoagulant activity (Thaler).

Procoagulant properties of tumor cell MP have been an area of intense study. A range of endothelial, monocyte and leukocyte MP along with tissue factor bearing MP appear to have a coagulant potential and have shown to be elevated in various such as cancers such as pancreatic, breast and prostate (Pilzer*, et al* 2005, Simak and Gelderman 2006).

A recent in vivo live microscopy mouse model with pancreatic cancer demonstrated that TF bearing MP released from the cancer cells entered circulation and participated in the thrombus formation at a distant site (Thomas, 2009).

The most important evidence for role of MP in VTE and cancer comes from clinical studies showing increased numbers and procoagulant activity of MP in cancer (Langer). Elevated levels of tissue factor bearing MP were associated with VTE events in those with advanced malignancy particularly pancreatic cancer. The microparticle levels in cancer patients also predicted the development of thrombosis, with the one year estimate of those with TF

Microparticles: Role in Haemostasis and Venous Thromboembolism 11

There are several approaches to detection and measurement of MP. The methods are usually based on the ability of the assay to either enumerate or assess functional activity of the MP.

Most of the assays under this section relate to either the prothrombotic function of MP or measuring the phospholipid content of MP. This can be done in the liquid phase e.g. a clot based assay such as the XACT test or by estimation of prothrombinase activity using an ELISA (Exner, 2003). The advantages of these approaches are that they provide an indication of the procoagulant activity of MP. The drawback is that the cell of origin for the MP cannot

Flow cytometry is the most widely employed quantitative technique. The gating of small particles continues to be a challenge but flow cytomtery continues to be the only robust technique which can demonstrate the cell of origin for the MP. This is an important asset of flow cytometry. However, there is significant variability amongst flow cytometers and the ISTH subcommittee on vascular biology recently conducted a workshop on standardization of MP by flow cytometry (Lacroix). It remains a popular approach for detection of MP for the following reasons:1)Rapid turn around time 2)Both fresh and frozen specimens may be used 3)The expression of two or more antigens on the MP may be simultaneously

However it has the following drawbacks: 1) The detection of particles less than 0.3µm is difficult by flow cytometry as the detection is limited by particle size in the same order of magnitude wavelength of the laser ( about 488 nm 2) Different machines have different sensitivities 3) It is difficult to automate 4) Centrifugation speeds for sample processing are variable and not standardized (Freyssinet, 2005). Several new approaches to flow cytometry include using impedance flow cytometry and using Raman microspectrophotometry effect

The capture of MP into immobilized annexin V or cell specific antibodies using an ELISA based assay have ben the other major approaches (Enjeti, 2007). Solid phase assays have the advantage of picking up microparticles irrespective of size. However interference of soluble antigens, variable quality of antibodies used for antigen capture and non-exclusion of

In the recent years there has been an adaptation of nanoscale technologies such as atomic force microscopy and nanoparticle measurement techniques. These methods claim to accurately measure particles in the nanoscale size range (Yuana). For example , one such nanoscale technique uses the brownian motion of these small particles to detect and measure them (Harrison, 2009). These methods are expensive, intensive to perform and not yet widely available (Lawrie, 2009). Moreover, the clinical utility of such techniques is not yet established. Recently a proteomic approach to analysis of MP has been described,

demonstrated 4)Easy method for quantification using commercial beads.

to cover the size and particle discrimination issues (Ayers, 2011).

microsomes are some of the disadvantages.

**4.3 Nanoscale and newer technologies** 

**4. Measuring microparticles** 

**4.1 Functional assays** 

**4.2 Quantitative assays** 

be determined.

bearing MP being about 34% (Thaler, 2011). In contrast those who did not develop thrombosis did not have a detectable level of tissue factor bearing microparticles.

## **3.1.4 Disease groups associated with venous or arterial thrombosis**

There are a number of conditions associated with elevated MP. Most of these disease states are associated with an increased risk of thrombosis. They essentially seem to reflect the health and pathophysiology of the endovascular system. Table 3 below gives a list of conditions where they have been found to be elevated.


Table 3. List of conditions associated with thrombosis and elevated MPs in circulation.

#### **3.2 Microparticles and atherothrombosis**

The role of MP in promoting atherothrombosis has also been another area of study (Cimmino). In one report, shed membrane microparticles were seen to be produced in human atherosclerotic plaques and were a critical determinant of thrombogenecity after plaque rupture (Mallat, 1999). The apoptosis occurring after plaque disruption or rupture was closely associated with TF expression on cell membranes leading to thrombogenecity. These MP were observed to express phosphotidylserine and some expressed CD11a which is an adhesion molecule (Martinez, 2005) (Morel, 2006). Given the links between inflammation and thrombosis, the emerging role of MP in atherothrombosis is not surprising (McGregor, 2006; Meerarani, 2007).
