Background: Gender-related differences in incidence of arterial thrombosis have been a focus of interest for years. The platelet integrin IIb 3 is primarily responsible for the interaction between platelets and fibrinogen and consecutive thrombus growth. In this study, we evaluated platelet adhesion onto immobilized fibrinogen under venous and arterial flow conditions in men and women. Methods: Platelets in whole anticoagulated blood were labelled with the fluorescence dye Mepacrine and perfused through the rectangular flow chamber over glass cover slips coated with fibrinogen (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 seconds, 1 and 5 minutes after the start of perfusion. Results: During perfusion, the platelet adhesion linearly increased in regard to exposition time and shear rate. After five minutes of perfusion the platelet adhesion onto immobilized fibrinogen showed no significant gender related difference, neither at 50 s-1 nor at 500 s-1 and 1500 s-1 (p > 0.05), respectively. No significant difference in platelet adhesion onto immobilized fibrinogen, in regard to the menopausal status, was either observed (p > 0.05). Conclusion: In our in vitro experimental system, hormonal differences between men and women did not influence platelet adhesion onto immobilized fibrinogen, neither under venous nor under arterial rheological conditions.

Evaluation of the TEG platelet mapping assay in blood donors
Background: Monitoring of antiplatelet therapy in patients at cardiovascular risk is difficult because existing platelet function tests are too sophisticated for clinical routine. The whole blood TEG Platelet Mapping assay measures clot strength as maximal amplitude (MA) and enables for quantification of platelet function, including the contribution of the adenosine diphosphate (ADP) and thromboxane A2 (TxA2) receptors to clot formation. Methods: In 43 healthy blood donors, the analytical (CVa) and inter-individual variability (CVg) of the TEG Platelet Mapping assay were determined together with platelet receptor inhibition in response to arachidonic acid (AA) and ADP. Results: The CVa of the assay for maximal platelet contribution to clot strength (MAThrombin) was 3.5%, for the fibrin contribution to clot strength (MAFibrin) 5.2%, for MAAA 4.5% and for MAADP it was 6.6%. The MAThrombin CVg was 2.8%, MAFibrin 4.7%, MAAA 6.6% and for MAADP it was 26.2%. Females had a higher MAThrombin compared to males (62.8 vs. 58.4 mm, p = 0.005). The platelet TxA2 receptor inhibition was 1.2% (range 0 10%) and lower than for the ADP receptor (18.6% (0 58%); p < 0.0001). Conclusion: The high variability in ADP receptor inhibition may explain both the differences in response to ADP receptor inhibitor therapy and why major bleeding sometimes develops during surgery in patients not treated with ADP receptor inhibitors. An analytical variation of ~5 % for the TEG enables, however, for routine monitoring of the variability in ADP receptor inhibition and of antiplatelet therapy.

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Clopidogrel in Orthopaedic patients: a review of current practice in Scotland
Background: Clopidogrel bisulfate is an antiplatelet agent used to prevent ischaemic events in patients with vascular disease. Current guidelines recommend withholding clopidogrel for 7 days pre-operatively. However these are not based on orthopaedic patients. We therefore decided to survey current orthopaedic practice to see whether this complied with available clinical data.MethodA questionnaire was sent to all orthopaedic consultants in Scotland.Four haematology departments, and the manufacturers, were contacted to ask for their recommendations, and a database search was performed. Results: 140 questionnaires were sent with a 60.7% response. 84.7% of respondents have encountered patients on clopidogrel. Of those, 13.9% did not routinely stop it, and 86.1% stopped it 5 21 days pre-operatively (47.2% at 7 days).45.9% had a unit policy on stopping clopidogrel, and the majority (69.4%) did not consult their haematology department prior to instituting their policy.Increased peri-operative bleeding was the most reported complication (22.6%). However this was only noted in those who stopped clopidogrel greater-than 7 days pre-operatively.Haematology advice ranged from continuing clopidogrel peri-operatively to stopping it 7 days pre-operatively and starting low-molecular-weight-heparin for thrombo-prophylaxis. The manufacturers suggested stopping clopidogrel 7 days pre-operatively. An internet search did not reveal any data on the effect of clopidogrel peri-operatively in orthopaedic patients.DiscussionRecommendations on stopping clopidogrel have evolved from studies conducted on patients undergoing cardio-thoracic surgery. There is no data available on the effect of clopidogrel in orthopaedic practice. Our survey indicates that increased bleeding has not been found in patients who continue clopidogrel peri-operatively.Almost half of respondents complied with current recommendations, stopping clopidogrel 7 days pre-operatively. However there remains a lack of consensus amongst orthopaedic surgeons.Currently elective patients should stop clopidogrel 7 days pre-operatively, and emergency patients should stop clopidogrel on admission, however their operation should not be delayed due to clopidogrel usage.

HPA-1 polymorphism of IIb 3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system
Background: Platelet adhesion and subsequent thrombus formation on a subendothelial matrix at the site of vascular damage play a crucial role in the arrest of posttraumatic bleeding but also in different pathological thrombotic events, such as acute coronary syndrome and stroke. Recently published studies have clearly demonstrated that platelet integri IIb 3 is intimately involved in the occlusive thrombus formation at the site of endothelial damage. Therefore, any genetic variation in the expression of this receptor may lead to an excessive bleeding or excessive thrombus formation. In this study, we evaluated the influence of HPA-1 polymorphism of integrin IIb 3 on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating blood flow. Methods: Platelets in anticoagulated whole blood [49 healthy previously genotyped blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 sec, 1 and 5 minutes after start of perfusion. Results: During perfusion, the platelet adhesion linearly increased with regard to exposition time and shear rate. Perfusion of blood preincubated with Abciximab over fibrinogen-coated cover-slips showed reduced platelet adherence (absolute fluorescence: 168 35 U vs. 53000 19000 at control experiments, p < 0.05), as well as by perfusion over BSA-coated glass coverslips. Platelet with HPA-1a/1a genotype exhibited initial better adhesion but they also exhibited higher detachment under arterial flow conditions compared to the HPA-1b/1b platelets. Analysis of stable adhesion rate indicate that the platelets carrying the HPA-1b/1b genotype have a higher reactivity threshold for initial interaction with fibrinogen but under the higher shear rate (in regard to time of perfusion) also realize more stable bonds with fibrinogen than platelets with the HPA-1a/1a genotype. Conclusion: Our data support the contention that genetically determined variants of platelet integrins IIb 3 could play a role in arterial thrombogenesis and thus confirm the hypothesis derived from epidemiological studies.

Hyperhomocysteinemia in women with unexplained sterility or recurrent early pregnancy loss from Southern Italy: a preliminary report
Background: Hyperhomocysteinemia has been described as a risk factor for unexplained recurrent pregnancy loss. Increased levels of homocysteine may be due to inadequate dietary intake of folate and vitamin B12 and inherited defects within the methionine-homocysteine pathway such as MTHFR C677T gene polymorphism. However, the association between hyperhomocysteinemia and sterility problems have been underlined only for recurrent pregnancy loss while a relationship between hyperhomocysteinemia and female sterility is still matter of discussion.AimThis study sought to find out a possible relationship between sterility (primary sterility or secondary sterility due to recurrent pregnancy loss) and homocysteine metabolism.Patients and MethodsWe selected 20 patients with recurrent pregnancy loss, 20 patients with unexplained female sterility and 20 healthy women as control group. Several whole blood samples were collected by venipuncture. Firstly homocysteinemia and other related variables were tested (i.e. folate and vitamin B12 levels); thereafter DNA was extracted by a further whole blood sample collected in EDTA in order to screen MTHFR C677T gene polymorphism. Statistical analysis was performed by chi square test; differences were considered to be significant if p < 0.05. Results: The median fasting total plasma homocysteine concentration was 19.2 6.14 M for patients with recurrent pregnancy loss, while was 21.05 8.78 M for patients with unexplained sterility, vs 7.85 3.31 M of control group (p < 0.05). Fifteen patients with unexplained female sterility showed MTHFR C677T homozigosity vs 17 with recurrent pregnancy loss and 3 in the control group (p < 0.05). On the other hand no significant differences were found in the levels of vitamin B 12 in the three groups, while reduced folate concentrations were found in women with unexplained female sterility and recurrent pregnancy loss (p < 0.05 vs control group.DiscussionMTHFR C677T gene polymorphism is frequent in the studied populations. These data raise questions on the role of the homocysteine metabolism in sterility problems. Even though increased homocysteine (i.e. > 15 M) and MTHFR C677T homozigosity have already been described as risk factors for recurrent pregnancy loss, few studies evaluated their role in women with unexplained sterility. Further studies on larger series are needed to better understand the role of homocysteine metabolism, including folate metabolism, in this clinical setting.

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