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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation Introduction and aim: Mimics of mast cell-derived heparin proteoglycans (HEP-PG) can be tailored to molecules carrying both antiplatelet and anticoagulant properties. These dual antiplatelet and anticoagulant (APAC) constructs can also shield adhesion molecules such as P-selectin and VCAM-1 expressed by endothelial cells upon atherosclerosis development. We hypothesize that via this way, APAC prevents macrophage accumulation and lesion development. In this study, we therefore determined the efficacy of APAC in inhibiting atherosclerosis. Methods Male western-type diet fed apoE-/- mice were equipped with perivascular carotid artery collars to induce atherosclerosis. In this collar model, mRNA expression of adhesion molecules such as ICAM-1, VCAM-1, P-Selectin but also of Platelet Factor 4 (PF4) are significantly upregulated upon lesion development (all P Results APAC treatment did not affect body weight or plasma total cholesterol levels of the mice during the experiment. Interestingly, carotid artery plaque size was reduced by over 50% upon APAC treatment (APAC: 50±10*10E3 versus controls: 102±13*10E3 square µm; P Conclusion We here show that APAC effectively inhibits atherosclerotic lesion development when administered during lesion initiation and may have potential as therapeutic agent to prevent atherosclerosis.

A survey of 21 haemophilia doctors, throughout Europe, who care for a total of approximately 5000 patients with bleeding disorders addressing practice and opinions regarding prophylaxis in patients aged 16-24 years and adults aged over 50 years, is presented. The outcome of adolescent patients who reduced or stopped prophylaxis was recorded. Eighteen of 19 respondents would consider modification of established prophylaxis in the adolescent age group, principal considerations being avoidance of risks of further concentrate exposure, predicted poor compliance and treatment costs. The preferred age for modification was 16-20 years, but there was very little consensus on the particular prophylactic regime recommended. Approximately, half of a cohort of 218 patients with severe haemophilia successfully reduced or stopped prophylaxis when they reached adolescence. Only 26 of 92 (28%) of the patient cohort who stopped prophylaxis, required reintroduction of a prophylactic regime and 12 of 59 (20%) of those who reduced the intensity of prophylaxis had to reintroduce a more intensive regime. A majority of respondents would consider starting prophylaxis in those over 50 years. There was no consensus as to indications for this practice or the nature of the prophylaxis protocol. We conclude that there is an absence of consensus on the management of patients with severe haemophilia, as they pass through adolescence and young adulthood, and reach the age of 50. Aggregate outcome data suggest a significant proportion of patients in the 18-22 years age range may be able to reduce or stop prophylaxis. A substantial number of older patients are on prophylaxis.

Hemostatic variables and platelet function were assessed as a part of a genetic study in 15 patients with symptomatic peripheral arterial occlusive disease (PAOD) and 15 healthy siblings from ten families. D-dimer, a degradation product of cross-linked fibrin, was increased in the PAOD group (mean +/- SD) (448 +/- 177 vs. 333 +/- 121 ng/ml, p0.05). Ristocetin-induced maximal platelet aggregation (RIPA) was reduced in the PAOD group in response to both a higher (0.75 mg/ml) (67 +/- 28 vs. 87 +/- 14%, p = 0.02) and a lower (0.55 or 0.60 mg/ml) (33 +/- 21 vs. 59 +/- 32%, p = 0.02) concentration of ristocetin. Accordingly, the rate of primary aggregation was smaller, and a larger threshold concentration of ristocetin was needed to cause aggregation. However, ristocetin cofactor activity, von Willebrand factor (vWF) antigen and its multimer distribution, plasma glycocalicin, platelet glycoprotein Ib content and the binding of vWF to frozen and thawed washed platelets were equal in both groups. Thus, the observed reduced RIPA in patients with PAOD is less likely to reflect a down-regulation or blunted binding affinity in the platelet surface glycoprotein Ib.

Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI.