Graham J. Caine

Platelet activation, coagulation and angiogenesis in breast and prostate carcinoma

In health, haemostasis and angiogenesis are tightly regulated processes, but may become deregulated in cancer. Recent evidence suggests that platelet activation may link these processes as platelets can release angiogenic factors such as vascular endothelial growth factor (VEGF). Furthermore, inflammation has also been implicated in regulating both coagulation and angiogenesis, possibly by activating platelets directly and increasing, for example, plasma fibrinogen. We hypothesized relationships between plasma markers of the processes in two common forms of cancer. Plasma levels of VEGF (reflecting angiogenesis), soluble P-selectin, (marking platelet activation), tissue factor [TF], fibrinogen and fibrin D-dimer (coagulation markers), and serum levels of IL-6 (inflammation) were measured by ELISA in 30 patients with biopsy-proven breast cancer, 30 patients with biopsy-proven prostate cancer, and 30 age- and sex-matched controls for each group. Prostate specific antigen was also measured in the men. Release of VEGF from IL-6 stimulated platelets was assessed by ELISA. Plasma levels of IL-6 (P <0.02), VEGF, soluble P-selectin, fibrinogen, and fibrin D-dimer (all p <0.01) were significantly raised in breast cancer, whereas VEGF, soluble P-selectin, fibrin D-dimer (all p <0.01) and fibrinogen (p <0.05) were significantly raised in prostate cancer. Significant correlations were found between IL-6 and VEGF (p <0.01), and IL-6 and soluble P-selectin (p = 0.038) in breast cancer. Further experiments demonstrated an in vitro IL-6 induced dose-dependent release of VEGF from platelets. In conclusion, strong relationships between IL6 and VEGF, but not with coagulation or platelet markers, and release of VEGF from IL-6 stimulated platelets, suggest a role for inflammation and platelets in angiogenesis.

Platelet P-Selectin Levels in Relation to Plasma Soluble P-Selectin and β-Thromboglobulin Levels in Atrial Fibrillation

Background and Purpose— The increased risk of stroke and thromboembolism in atrial fibrillation (AF) may be related to a prothrombotic or hypercoagulable state, with abnormalities of hemostasis and platelet activation. To investigate the role of platelets in AF and the influence of antithrombotic therapy, we developed and then applied a new assay to detect the absolute amount of P-selectin per platelet (pP-selectin) based on cell lysis. Thus, pP-selectin in AF patients was compared with that of healthy controls and also with plasma soluble P-selectin (sP-selectin) and &bgr;-thromboglobulin as established indices of platelet activation. Methodsmdash; We studied 122 patients (mean [SD] age, 71 [9] years; 65 men) with chronic AF of >6 weeks’ duration: 34 were not on antithrombotic therapy, 30 were taking aspirin (75 to 300 mg/d), and 58 were fully anticoagulated with warfarin. pP-selectin was compared with sP-selectin and plasma &bgr;-thromboglobulin levels (enzyme-linked immunosorbent assay). Results were compared with those of 23 healthy controls (mean [SD] age, 74 [9] years; 7 men) in sinus rhythm. Results— pP-selectin was significantly lower in AF patients on no antithrombotic therapy (P ∓0.03) than in healthy controls, but sP-selectin and &bgr;-thromboglobulin levels were not significantly different and did not differ in patients taking aspirin or warfarin. However, pP-selectin was lower in patients with AF on aspirin than in those on warfarin (P <0.05). pP-selectin/sP-selectin correlated significantly in healthy controls (r ∓0.47, P ∓0.03) but inversely (r ∓−0.43, P ∓0.03) in AF patients on no antithrombotic therapy. Conclusions— Lower levels of pP-selectin may represent a depletion of pP-selectin after platelet activation in AF. Aspirin further decreases pP-selectin levels compared with warfarin. On the basis of the principle of platelet lysis, we demonstrate that it is possible to determine the amount of P-selectin per platelet, which may be regulated in the megakaryocyte through a cyclooxygenase-dependent pathway.

Platelet-derived VEGF, Flt-1, angiopoietin-1 and P-selectin in breast and prostate cancer: further evidence for a role of platelets in tumour angiogenesis.

BACKGROUND: Cancer is a complex multi‐factorial disorder that may commonly show abnormal angiogenesis in such patients. Recently, platelets have been postulated to have a major role in both these processes, suggesting that anti‐platelet strategies may be useful in cancer treatment. MATERIALS AND METHODS: To further investigate the role of platelets in angiogenesis, we used a novel platelet lysate assay to analyse platelet contents in breast cancer (n = 30) and prostate cancer (n = 30) patients and age‐ and sex‐matched controls (n = 60). Markers of angiogenesis (vascular endothelial growth factor (VEGF), angiopoietin‐1 and–2 (Ang‐1, ‐2), and their respective receptors (Flt‐1 and Tie‐2) plus a marker of platelet activation (P‐selectin (P‐sel)), were all measured in platelet lysate by enzyme‐linked immunsorbent assay. RESULTS: Platelet lysate from breast cancer patients contained higher levels of VEGF (P < 0.0001), Ang‐1 (P = 0.0186) and P‐sel (P = 0.0002), compared to healthy controls. Platelet lysate from prostate cancer patients had elevated VEGF (P = 0.008) but not Ang‐1 or P‐sel. There were no significant differences between levels of Flt‐1 between patients and controls, and both Ang‐2 and Tie‐2 were undetectable in both patient groups and control platelet lysate. CONCLUSION: We have shown that our previously developed platelet lysate technique could be used to measure indices of angiogenesis, and their respective receptors, and that this assay can be applied to patients with cancer. Our study also provides further evidence that platelets may influence angiogenic abnormalities in human cancer. The platelet may be a useful target in anti‐cancer strategies.

Soluble p-selectin should be measured in citrated plasma, not in serum.

hood. The patients with splice site mutations had the highest incidence of lymphomas (seven of 58 kindred or 12Æ1%). For intron 6 splice site mutations, the incidence was four of nine kindred (44%, statistically different from the incidence for all mutations, v test, P < 0Æ0002). For the single mutation intron 6 (+ 5), g fi a, the incidence was three cases in seven kindred. We previously demonstrated the discordant expression of WASP in blood cells of five patients with two intron 6 mutations (Shcherbina et al, 1999). Whereas T cells of these patients had low but readily detectable levels of WASP ( 10–20% of normal levels), the patients’ peripheral B cells, as well as their Epstein–Barr virus-transformed B-cell lines, did not express the protein. Similar discordance between T and B cells was also demonstrated for a patient with an exon 1 nonsense mutation, who died of lymphoma, and two members of a family with an intron 7 splice site mutation, who have mild disease. We propose that discordant expression of WASP in T and B cells is an indication of increased risk of lymphomas in patients, even for those with an otherwise mild course of the disease. Bone marrow transplantation may be indicated for this subgroup of mild WAS patients.

Platelet adhesion in hypertension: Application of a novel assay of platelet adhesion

Background. The increased risk of thromboembolism in hypertension may be related to a prothrombotic or hypercoagulable state, with abnormalities in haemostasis and platelet function. Objective. To investigate the role of platelets in the pathogenesis of thrombosis in hypertension, we applied a novel new assay to detect and quantify the degree of platelet adhesion to a defined coagulation molecule. Patients and methods. Platelet‐rich plasma (PRP) and citrated plasma (CP) were obtained from 50 patients with hypertension (25 treated, and 25 untreated) and 30 healthy controls. A suspension of 2×107 platelets were incubated for one hour in microtitre plates pre‐coated with 5mg/mL fibrinogen. The supernatant was carefully aspirated, lysed with 5% tween and stored at −70°C as supernatant platelet lysate (SPL). The wells were carefully washed with saline and bound platelets lysed as before, and stored at −70°C as bound‐platelet lysate (BPL). Soluble P‐selectin (sP‐sel) was determined in CP, SPL and BPL by enzyme‐linked immunosorbent assay (ELISA). Results. Patients with hypertension (both treated and previously untreated) had increased platelet adhesion, as determined by increased lysate sP‐sel (P = 0.002) in BPL, with no change in SPL (P = 0.5) compared to healthy controls. There was no significant difference between treated and previously untreated hypertensives. Conclusion. Platelets from patients with hypertension display increased adhesion to an important coagulation factor (fibrinogen). This may, in part, account for the increased risk of thrombosis seen in these patients.

Platelet adhesion in breast cancer: development and application of a novel assay.

The increased risk of thromboembolism in cancer may be related to a prothrombotic or hypercoagulable state, with abnormalities of haemostasis and platelet activation. To further investigate the role of platelets in this disease, we developed and applied a new assay to detect and quantify platelet adhesion to the well-defined subendothelial substrate, fibrinogen. Platelet-rich plasma was obtained from 31 females with breast cancer (13 metastatic, 18 benign), and 30 healthy female controls, re-suspended to 2 × 108 cells/ml and 100 μl and incubated for 1 h in microtitre plates pre-coated with fibrinogen (5 mg/ml). The supernatant was carefully aspirated, lysed with Triton X-100 and stored at −70°C as supernatant-platelet lysate. The microtitre wells were carefully washed with saline, bound platelets lysed with Triton, and the lysate stored at −70°C as bound-platelet lysate. P-selectin was determined in supernatant-platelet lysate and bound-platelet lysate for each patient by enzyme-linked immunosorbent assay. Interpreting differences in P-selectin in different lysates as reflective of adhesion, patients with cancer had increased platelet adhesion (absolute and percentage, both P < 0.001) compared with healthy controls. There was also more adhesion (P < 0.001) in metastatic disease compared with non-metastatic disease. Patients with breast carcinomas, and, in particular, those with metastatic disease, have a higher degree of platelet adhesion, which may by quantified by a novel method based on cell lysis. This increase in platelet adhesiveness may be related to an increased risk of thromboembolism in these patients.

A comparison of plasma versus histologic indices of angiogenic markers in breast cancer.

BackgroundOver-expression of angiogenic growth factors and their receptors, and high levels of these molecules in the blood, are a common feature of cancer although the relationships between cell expression and plasma levels are unknown. We hypothesized a significant correlation between the expression and cellular distribution of vascular endothelial growth factor (VEGF), its receptor Flt-1, and the angiopoietin receptor Tie-2 with levels of these molecules in the plasma. MethodsThe tissue expression of VEGF, Flt-1, and Tie-2 were investigated by immunohistochemistry, and plasma levels assessed by enzyme-linked immunosorbent assay in 36 patients with breast cancer and 15 with benign breast disease. ResultsDespite expected significant differences in plasma levels of the molecules (P<0.03 to <0.001), no significant differences were found in Tie-2, VEGF, and Flt-1 tissue expression between breast cancer and benign disease controls. No significant correlations were observed between plasma levels of their tissue expression. ConclusionsTissue expression of Tie-2, VEGF, and Flt-1 may not be an overly sensitive tool for assessing abnormalities of coagulation, platelet activation, and angiogenesis in human cancer. Plasma markers may not be representative of tumor activity, and may not come wholly from tumor cells. Instead these markers may be indicative of endothelial dysfunction in cancer patients.

Abnormal vascular, platelet and coagulation markers in primary thrombocythaemia are not reversed by treatments that reduce the platelet count

Although the primary aim of the treatment of primary thrombocythaemia is to reduce platelet counts, its effect on platelet activation, vascular dysfunction and coagulopathy, which are also abnormal, is unknown. We therefore hypothesised that successful treatment of primary thrombocythaemia is accompanied by an improvement in markers of these processes. To test this hypothesis, we compared 38 patients with 15 healthy age and sex-matched controls. Seven untreated patients had platelet counts higher than those 31 on treatment, that were in turn higher than the 15 controls (all p<0.01). Plasma fibrinogen, P-selectin, von Willebrand factor and sVCAM were higher in both patient groups compared to controls (all p<0.05), but not between patient groups. sE-selectin and sICAM were unaltered between the three groups. D-dimers were higher in treated patients than in controls. We conclude that successful thrombocytosis treatment does not normalise markers of coagulation, platelet, or endothelial pathophysiology. This may account for continuing risk of pathology in those whose platelet counts have been normalised.

Vascular endothelial growth factor and platelets in primary (essential) thrombocythaemia

Primary or essential thrombocythaemia (PT), a rare myeloproliferative disorder, is characterised by high platelet numbers, an increased risk of thrombosis and haemorrhage, and a reduced life expectancy. In addition to thrombocytosis, additional platelet abnormalities include alterations in soluble P-selectin (sP-selectin), platelet P-selectin (pP-selectin) and platelet factor 4 (Musolino et al, 2000; Villmow et al, 2002). Vascular endothelial growth factor (VEGF) is an angiogenic product of several cells, including endothelial cells and platelets (Blann et al, 2004), and increased levels (thereby possibly implying alterations in angiogenesis), are present in PT and other myeloproliferative disorders (Di Raimondo et al, 2001; Musolino et al, 2002). The implications of this finding are unknown. Furthermore, the source of this VEGF is unclear and could have pathophysiological and/or therapeutic connotations. We therefore set out to test the hypothesis that platelets are a major source of plasma VEGF in patients with PT. To do this we measured levels of VEGF in and plasma and platelet lysates in a simple cross-sectional study comparing patients to healthy ageand sex-matched controls, and compared levels to those of sP-selectin. This study was a sub-analysis of a group of patients previously studied (Blann et al, 2004). Patients were recruited if they fulfilled diagnostic criteria of PT. Almost all were being treated with combinations of aspirin (75 mg/d) and anagrelide (mean ± SD 1Æ6 ± 0Æ6 mg/d) or hydroxycarbimide (0Æ95 ± 0Æ3 g/d). Patients were compared with 15 ageand sexmatched healthy controls recruited from hospital staff. All subjects gave written informed consent and the study had the approval of the Ethics Committee of City Hospital, Birmingham, UK. In the routine Haematology laboratory, EDTAplasma was tested for platelet count (Advia, Bayer, Newbury, UK). In the research laboratory, VEGF and sP-selectin were measured in citrated plasma by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Abingdon, UK). The total mass of VEGF per platelet was estimated by lysing a set number of thrice-washed platelets with 0Æ5% Triton X-100 (Sigma, Poole, UK), and then measuring VEGF in the lysate by ELISA to give the mean mass of P-selectin per platelet (Kamath et al, 2002). The results are shown in Table I. As expected (Musolino et al, 2000; Villmow et al, 2002; Blann et al, 2004), patients had a higher platelet count, sP-selectin and VEGF than controls. However, there was no difference in the mass of VEGF per platelet. Within the patient group, there were no significant Spearman’s correlations between the platelet count and VEGF (r 1⁄4 )0Æ059, P 1⁄4 0Æ752), nor between sP-selectin and VEGF (r 1⁄4 0Æ081, P 1⁄4 0Æ659). However, the platelet count correlated with sP-selectin (r 1⁄4 0Æ373, P 1⁄4 0Æ036). Increased levels of plasma VEGF in PT are an established finding and the presence of this angiogenic growth factor in platelets naturally leads to the question of whether or not this is important. Interest lies in the possibility that platelet VEGF may contribute to angiogenesis within the bone marrow and thus the disease process. The observations of the present study suggest this may not be the case. First, despite differences in plasma levels, there was no difference in platelet VEGF between the patients and matched controls. Secondly, neither platelet nor plasma VEGF correlated significantly with either the platelet count, or with sP-selectin. We therefore suggest that raised plasma VEGF in PT is not the direct consequence of altered platelet activity.