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Objective: The purpose of this study was to measure the in vivo platelet activation and thrombin generation in arterial blood after passing a subintimal conduit., Methods: Subintimal percutaneous transluminal angioplasty (SPTA) is a technique where a subintimal channel is created, allowing recanalization of long peripheral arterial occlusion. From 10 patients with intermittent claudication, undergoing successful SPTA for femoropopliteal occlusive disease, we collected antecubital venous blood samples immediately before treatment, preprocedural arterial blood samples taken at the entry level proximal to the vessel occlusion, and subsequently at the reentry level after successful recanalization. Venous follow-up blood samples were taken after 24 hours. Plasma concentrations of β-thromboglobulin (β-TG), RANTES, and Prothrombin fragment (F1 + 2), were determined by immunoassay. Fibrinogen binding to platelets, leukocyte-platelet adhesion, and P-selectin were determined by flow cytometry., Results: We found a statistically significant transluminal increase in the plasma concentrations of RANTES, β-TG and F1 + 2 (p = 0.002, 0.001 and 0.001 respectively), which all normalized within 24 hours. Platelet-leukocyte aggregates significantly decreased after 24 hours compared with preprocedural and preentry levels (3.26% versus 5.26 %, p = 0.017). P-selectin expression on circulating platelets was statistically significantly increased in the blood sample taken at the re-entry level compared with the pre-procedural and pre-entry level (p = 0.007). After 24 hours there was no statistically significant difference to pre-procedural levels. There was no significant change in platelet fibrinogen binding at any levels., Conclusion: When passing a subintimal conduit, in vivo sampled blood demonstrated an extremely rapid and substantial uniform platelet activation and thrombin generation.

Platelet-derived microparticles (PMPs) are considered a marker of platelet activation. They vary considerably in size, and flow cytometry, the predominant method used to assay PMPs, is only detecting larger PMPs (>0.1 microm). We describe here a method that quantifies the amount of PMP-located GPIIb antigen in detergent-treated platelet-free plasma (PPP) by means of a one-step time-resolved immunofluorometric assay (TR-IFMA). This assay uses a streptavidin-coated microwell plate and two different monoclonal antibodies to GPIIb (CD41), one conjugated to biotin and the other labeled with europium ion. A wide linear range standard curve with low background and a high sensitivity was obtained. Pre-assay ultracentrifugation or filtration of PPP extensively reduced the fluorometric signal, indicating that the GPIIb antigen is mainly particle-located. A strong correlation between the amount of GPIIb and PMP as detected by flow cytometry was found. Consequently, the assay can be used to study PMP-related phenomena and, in contrast to flow cytometry, can be used on frozen samples and is independent of PMP size.

An inappropriate and persistent immune activation has been suggested to contribute to long-term mortality and morbidity after heart transplantation. Several lines of evidence suggest that platelets do not only promote thrombus formation, but also act as inflammatory cells. In the present study, we investigated if long-time survivors of heart transplantation (mean time since transplantation 6.5 yr) were characterized by enhanced platelet activation as assessed by different experimental approaches. Our main findings when comparing heart transplant recipients (n = 52) and age- and sex-matched healthy controls (n = 38) were: (i) platelets from heart transplant recipients showed enhanced expression of both P-selectin and CD63 as assessed by flow cytometry; (ii) platelets from these patients also contained significantly increased levels of soluble CD40 ligand and tended to release higher levels of this cytokine upon SFLLRN stimulation as assessed by enzyme immunoassay; (iii) heart transplant recipients had increased levels of soluble P-selectin in platelet-free plasma; and (iv) the enhanced platelet activation after heart transplantation was most pronounced in those with concomitant hypertension. These findings suggest that long-term survivors of heart transplantation are characterized by enhanced activation of platelets, possibly contributing to the persistent immune activation and clinical complications in these patients.