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Background:  For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy., Methods and Results:  To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention., Conclusion:  Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation., Competing Interests: None declared., (Thieme. All rights reserved.)

The formation of leukocyte-platelet aggregates (LPA), through the P-selectin - P-selectin glycoprotein ligand (PSGL)-1 axis, plays a pivotal role in atherothrombosis. In order to investigate the influence of platelet (pP-selectin) and soluble P-selectin (sP-selectin), and of variations in the genes encoding for P-selectin (SELP) and PSGL-1 (SELPLG) on LPA formation, we assessed monocyte (MPA)- and neutrophil-platelet aggregates (NPA) as well as pP-selectin by flow cytometry in 263 patients undergoing angioplasty and stenting. sP-selectin was determined by ELISA, the SELP Pro715 allele and the SELPLG Ile62 allele were determined by allele specific PCR. The Pro715 allele was significantly associated with lower levels of in vivo pP-selectin and sP-selectin, while agonists´ inducible pP-selectin was not influenced by the Pro715 allele. PP-selectin was significantly associated with MPA and NPA formation. The in vivo formation of MPA and NPA depended to 19 % and 7.4 %, respectively, on in vivo pP-selectin, irrespective of the Pro715 allele and the Ile62 allele carrier status. TRAP-6 inducible MPA and NPA depended to 34 % and 27 %, respectively, on TRAP-6 inducible pP-selectin, but were independent of the Pro715 allele carrier status. Carriers of the Ile62 allele showed a stronger correlation between TRAP-6 inducible pP-selectin and TRAP-6 inducible MPA/NPA than non-carriers. Furthermore, TRAP-6 inducible NPA were higher in Ile62 allele carriers, which suggests higher thrombin sensitivity. In conclusion, our findings point to the significant role of pP-selectin for MPA and NPA formation, while other variables like sP-selectin, the SELP Pro715 allele and the SELPLG Ile62 allele have less influence.

ATP released by activated platelets can serve as a positive feedback machinery to amplify platelet responses by activating P2X1 receptors. It has, however, not been defined how P2X1 activities influence thromboxane A2 (TXA2)-stimulated platelet functional responses. Our aim was to elaborate the molecular mechanisms of P2X1 engagements in TXA2-induced platelet secretion and aggregation. P2X1 inhibition by 1 µM NF449 inhibited platelet P-selectin expression induced by a low concentration of the TXA2 analogue U46619 (0.3 µM) (32.0 ± 2.0% vs 43.4 ± 3.0%; n=5; p<0.05). p38 inhibition by SB203580, but not ERK inhibition by U0126, elicited a similar inhibition by NF499. The combination of NF449 and SB203580 provided, however, no additive effects. U46619-induced platelet aggregation was similarly decreased by NF449 and SB203580 alone or in combination, and by P2X1 pre-desensitisation with α,β-Me-ATP. U46619 caused rapid and reversible P2X1-dependent p38 phosphorylation. However, the P2X1-p38 pathway mainly enhanced mild platelet activation by U46619, because α,β-Me-ATP supplementation or p38 blockade had no effect on intense platelet activation induced by a higher concentration of U46619 (3 µM). In conclusion, P2X1 activation, via p38 signalling, potentiates platelet activation initiated by low doses of U46619. Hence, the P2X1-induced p38 signalling promotes more robust platelet activation in response to mild platelet stimuli.

Data linking the response to antiplatelet therapy with clinical outcomes after angioplasty and stenting for lower extremity artery disease (LEAD) are scarce. Moreover, associations of in vivo and thrombin-inducible platelet activation with the occurrence of adverse events have not been investigated in these patients, so far. We therefore assessed clinical outcomes and on-treatment platelet reactivity by four test systems in 108 patients receiving dual antiplatelet therapy after infrainguinal angioplasty and stenting for LEAD. Further, in vivo and thrombin receptor-activating peptide (TRAP)-6-inducible glycoprotein (GP) IIb/IIIa activation and P-selectin expression were measured as sensitive parameters of platelet activation. The primary endpoint was defined as the composite of atherothrombotic events and target vessel restenosis or reocclusion. Residual platelet reactivity to adenosine diphosphate and arachidonic acid was similar between patients without and with adverse outcomes within two-year follow-up (all p>0.05). Further, the occurrence of clinical endpoints did not differ significantly between patients without and with high on-treatment residual platelet reactivity by all test systems (all p>0.05). In contrast, in vivo and TRAP-6-inducible platelet activation were significantly more pronounced in patients with subsequent adverse events (all p<0.05), and high levels of platelet activation were independent predictors of the primary endpoint (adjusted hazard ratios: 3.5 for high in vivo activated GPIIb/IIIa, 2.9 for high TRAP-6-inducible activated GPIIb/IIIa, 2.3 for high in vivo P-selectin, and 3 for high TRAP-6-inducible P-selectin; all p<0.05). In conclusion, in vivo and protease-activated receptor-1-mediated platelet activation predict two-year clinical outcomes in stable patients undergoing angioplasty and stenting for LEAD.

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5'-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

P-selectin variant 715Pro is associated with lower concentrations of plasma P-selectin and reduced risk for thrombosis. We examined the influence of 715Pro on P-selectin synthesis, post-translational processing, surface expression and function using HEK293 cells, which do not express endogenous P-selectin. Mass spectrometry revealed that HEK293 cells produced recombinant P-selectin which has a glycosylation pattern comparable to platelet P-selectin. Compared to wild-type transfectants, 715Pro transfectants have ~50% less terminally glycosylated P-selectin and accumulate more immature P-selectin in Golgi. Following Brefeldin A treatment, the majority of 715Pro P-selectin is not modified by Golgi enzymes, while wild-type P-selectin undergoes complete modification. Flow cytometry revealed that 715Pro transfectants have ~20% less P-selectin on the cell surface compared to wild-type transfectants. Secretion of P-selectin by 715Pro transfectants was about 38% lower compared to wild-type transfectants. Binding of HL-60 cells to 715Pro transfectants was ~29% lower than to wild-type transfectants. This observation was confirmed by the presence of fewer platelet-monocyte aggregates (PMA) in the blood of healthy individuals and patients with angiographically proven atherosclerosis, carrying 715Pro P-selectin compared to individuals with wild-type P-selectin. We conclude that the 715Pro variant impairs terminal glycosylation of P-selectin in Golgi, leading to reduced amounts of mature P-selectin and subsequently less surface expression and secretion of P-selectin. The reduced surface expression of 715Pro P-selectin contributes to inefficient adhesion to HL-60 cells or monocytes.

Farnesyl pyrophosphate (FPP) is an intermediate in cholesterol biosynthesis, and it has also been reported to activate platelet LPA (lysophosphatidic acid) receptors. The aim of this study was to investigate the role of extracellular FPP in platelet aggregation. Human platelets were studied with light transmission aggregometry, flow cytometry and [³⁵S]GTPγS binding assays. As shown previously, FPP could potentiate LPA-stimulated shape change. Surprisingly, FPP also acted as a selective insurmountable antagonist to ADP-induced platelet aggregation. FPP inhibited ADP-induced expression of P-selectin and the activated glycoprotein (Gp)IIb/IIIa receptor. FPP blocked ADP-induced inhibition of cAMP accumulation and [³⁵S]GTPγS binding in platelets. In Chinese hamster ovary cells expressing the P2Y₁₂ receptor, FPP caused a rightward shift of the [³⁵S]GTPγS binding curve. In Sf9 insect cells expressing the human P2Y₁₂ receptor, FPP showed a concentration-dependent, although incomplete inhibition of [³H]PSB-0413 binding. Docking of FPP in a P2Y₁₂ receptor model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP. In conclusion, FPP is an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y₁₂ receptor. It could be an endogenous antithrombotic factor modulating the strong platelet aggregatory effects of ADP in a manner similar to the use of clopidogrel, prasugrel or ticagrelor in the treatment of ischaemic heart disease.

The role of platelets in the development of atherosclerosis and obesity-related prothrombotic state is still under investigation. In this cross-sectional cohort study, we measured the levels of different platelet activation markers and evaluated their relationship with carotid intima-media thickness (IMT) along with other atherosclerotic risk factors in obese patients with or without atherosclerotic co-morbidities. We enrolled 154 obese patients, including 98 with either hypertension, type 2 diabetes mellitus or dyslipidaemia, 56 without these co-morbidities and 62 age- and sex-matched healthy controls. Platelet P-selectin expression and the number of platelet-derived microparticles (PMPs) were measured by flow cytometry; soluble P-selectin levels were analysed by ELISA and Thr715Pro P-selectin polymorphism was determined by PCR-RFLP. Carotid IMT was examined by ultrasonography. The levels of platelet activation parameters were significantly elevated in all obese subjects with increased carotid IMT compared to healthy controls. There was no effect of Thr715Pro genotype on soluble P-selectin levels in obese individuals contrary to normal subjects. Significant and positive association was revealed between carotid IMT and platelet P-selectin (p<0.0001), soluble P-selectin (p=0.039) and PMP (p=0.0001) levels. After adjusting for multiple variables, independent association was found between soluble P-selectin and fibrinogen (p=0.007), PMP levels and body mass index (p<0.0001) as well as platelet P-selectin and carotid IMT (p=0.012) plus plasminogen activator inhibitor-1 (p=0.009). In conclusion, P-selectin and PMP levels showed positive associations with abnormal carotid IMT and other risk factors in obesity suggesting a critical role of enhanced platelet reactivity in atherosclerotic wall alteration.

Several rodent models have been used to study deep venous thrombosis (DVT). However, a model that generates consistent venous thrombi in the presence of continuous blood flow, to evaluate therapeutic agents for DVT, is not available. Mice used in the present study were wild-type C57BL/6 (WT), plasminogen activator inhibitor-1 (PAI-1) knock out (KO) and Delta Cytoplasmic Tail (DCT). An electrolytic inferior vena cava (IVC) model (EIM) was used. A 25G stainless-steel needle, attached to a silver coated copper wire electrode (anode), was inserted into the exposed caudal IVC. Another electrode (cathode) was placed subcutaneously. A current of 250 muAmps over 15 minutes was applied. Ultrasound imaging was used to demonstrate the presence of IVC blood flow. Analyses included measurement of plasma soluble P-selectin (sP-Sel), thrombus weight (TW), vein wall morphometrics, P-selectin and Von Willebrand factor (vWF) staining, transmission electron microscopy (TEM), scanning electron microscopy (SEM); and the effect of enoxaparin on TW was evaluated. A current of 250 muAmps over 15 minutes consistently promoted thrombus formation in the IVC. Plasma sP-Sel was decreased in PAI-1 KO and increased in DCT vs. WT (WT/PAI-1: p=0.003, WT/DCT: p=0.0002). Endothelial activation was demonstrated by SEM, TEM, P-selectin and vWF immunohistochemistry and confirmed by inflammatory cell counts. Ultrasound imaging demonstrated thrombus formation in the presence of blood flow. Enoxaparin significantly reduced the thrombus size by 61% in this model. This EIM closely mimics clinical venous disease and can be used to study endothelial cell activation, leukocyte migration, thrombogenesis and therapeutic applications in the presence of blood flow.

Essential thrombocythaemia (ET) and polycythaemia vera (PV) are characterised by a high incidence of thrombotic complications due to high-shear stress of the vessel wall, blood hyperviscosity and hypoxaemia, all factors responsible for chronic endothelial dysfunction and platelet and leukocyte activation. We evaluated the activation status of vascular cells in 18 consecutive ET and 14 PV patients by measuring the plasma levels of the nitric oxide derivatives (NOX) (i.e. nitrites and nitrates) and of soluble selectins of platelet (P-selectin), endothelial cell (P-selectin and E-selectin) and leukocyte (L-selectin) origin. The effect of hydroxyurea (HU) therapy on these parameters was also investigated. NOX were significantly (p<0.01) increased in ET patients treated with HU (11.5 +/- 2.6 nM) compared to non-HU treated ET (1.41 +/- 0.3 nM) and to controls (4.78 +/- 2.49 nM). Multivariate analysis confirmed HU therapy as an independent predictor of higher NOX levels in ET. In addition, NOX significantly correlated with haematocrit. Plasma P-selectin was significantly elevated in ET (350 +/- 40 ng/10(6) platelets) and PV (482 +/- 53 ng/10(6) platelets) patients compared to controls (120 +/- 8 ng/10(6) platelets). In PV, also E-selectin (23.8 +/- 4.2 ng/ml) was significantly increased compared to controls (11.2 +/- 1.1 ng/ml; p<0.01). P-selectin was significantly correlated to platelet (R=0.33; p=0.01) and leukocyte count (R=0.6; p=0.000), while E-selectin (R=0.34; p=.014) and sL-selectin (R=0.3; p=0.03) were correlated with leukocyte count only. In the multivariate analysis, NOX predicted increased levels of E-selectin in ET, but not in PV patients. Our data demonstrate that ET and PV are characterised by an altered pattern of soluble selectins and NOX. HU-mediated increase of NOX levels could represent an additional antithrombotic mechanism of this drug.

Platelets activated during experimental acute myocardial infarction (AMI) contribute to myocardial injury. This study aimed to investigate whether platelets from patients with AMI increase myocardial injury after ischaemia and reperfusion in isolated rat hearts and the modification of this effect by the P2Y12 receptor antagonist cangrelor and the glycoprotein IIb/IIIa receptor blocker abciximab. Isolated rat hearts were subjected to 40 minutes of global ischaemia and 60 minutes of reperfusion. Hearts (four simultaneous experiments per patient) were infused with platelets from nine AMI patients (seven thrombolysed, all on aspirin), untreated or incubated with 10 microM cangrelor or 5 microg/ml abciximab. Control experiments were performed using platelets from healthy volunteers and platelet-poor plasma. P-selectin expression on isolated platelets was higher in AMI patients than in controls and was not modified by the treatments. Control platelets or platelet-poor plasma did mild or no harm. In contrast, platelets from AMI patients significantly augmented myocardial injury, as demonstrated by worse left ventricular (LV) developed pressure, higher maximal LV end-diastolic pressure and coronary resistance, and greater lactate dehydrogenase release and infarct size. Both cangrelor and abciximab greatly attenuated these effects. In conclusion, activated platelets from AMI patients increase myocardial injury after ischaemia and reperfusion, and cangrelor and abciximab attenuate this effect. The results support the notion that very early antiplatelet treatment might reduce infarct size by direct effects on reperfused myocardium in these patients.

The thienopyridine P2Y12 receptor antagonists clopidogrel and prasugrel prevent arterial thrombosis and are routinely used following percutaneous coronary intervention. However, the optimal level of P2Y12 blockade to effectively inhibit platelet function is unknown. These studies utilised the active metabolite of prasugrel (R-138727) to achieve a range of P2Y12 blockade in vitro and assessed several aspects of platelet function. Blood from healthy volunteers was incubated with R-138727 (0-10 microM). P2Y12 receptor number was assessed using a 33P-2MeSADP binding assay. Platelet aggregation (PA) was measured by optical aggregometry with ADP 2-20 microM. VASP phosphorylation, annexin V binding, microparticle formation and P-selectin expression were assessed by flow cytometry. Increasing numbers of unblocked receptors were required for a sustained aggregation response with decreasing concentrations of ADP. A P2Y12 receptor blockade of 60-80% resulted in strong inhibition of final PA response, P-selectin expression, microparticle formation and vasodilator-stimulated phosphoprotein (VASP). PA induced by ADP 2 microM and P-selectin expression were particularly sensitive to low levels of receptor blockade whereas the VASP phosphorylation assay was relatively insensitive, requiring 60% receptor blockade to achieve substantial inhibition. Different assays varied in their ability to discriminate particular ranges of P2Y12 blockade and 80% or greater P2Y12 receptor blockade is required for consistently strong inhibition of several aspects of platelet function. These data guide the interpretation of results from different assays used to monitor the effects of P2Y12 receptor antagonists.

Inflammation and thrombosis, two processes influencing each other, are involved in the pathogenesis of cerebrovascular disease. We showed that in patients with acute ischaemic stroke circulating platelets are activated and exhausted. To identify whether activated haemostasis might be cause or effect, we investigated the role of leukocyte and platelet activation in patients with severe asymptomatic and symptomatic carotid artery disease. Flow cytometry analysis demonstrated that monocytes from symptomatic (acute stroke aetiology) and asymptomatic patients were highly activated, shown by significantly enhanced presentation of inflammatory markers CD11b and thrombospondin-1 (TSP-1) on the surface. Both correlated positively with monocyte-platelet association rate. However, increased monocyte activation and elevated levels of monocyte-platelet associates in asymptomatic patients were restricted to patients with echo-lucent plaques, providing a close link between monocyte activation and plaque morphology. Circulating single as well as monocyte-bound platelets from symptomatic patients showed significantly enhanced surface expression of P-selectin and TSP-1, whereas platelets from asymptomatic patients were not significantly activated. These results indicate that monocytes activated by inflammation rather than platelets might be the candidates to initiate platelet-monocyte rosetting during the pathogenesis of atherothrombotic cerebral ischaemia and that haemostasis might be activated secondarily by the first occurring inflammation.

Microparticles (MP) are lipid vesicles from platelets, leukocytes and endothelial cells that are involved in early thrombogenesis. We evaluated a detailed time-course analysis of MPs on thrombogenesis and the associated tissue factor (TF) activity in wild-type, in gene-deleted for E- and P-selectins and with high levels of P-selectin expression after the initiation of venous thrombosis in mice. Inferior vena cava (IVC) ligation was performed on C57BL/6 mice (n = 191, 59 = wild-type [WT], 55 = gene-deleted for E- and P - selectins [knock-outs, EPKO] and 77 = elevated levels of soluble P-selectin, named Delta Cytoplasmic Tail (DeltaCT). Animals were euthanised at various time points to assess MP production, origin and thrombus weight. MPs were re-injected into separate mice at concentrations of 80,000 and 160,000 units, as well as from different ages. In addition, MPs from thrombosed animals were pooled and TF activity quantitated using a chromogenic assay. Thrombus weight correlated negatively with MPs derived from leukocytes, and positively with MPs derived from platelets for WT animals (p < 0.05), while MPs from platelets presented a positive correlation to thrombus weight in the WT and EPKO groups (p < 0.01). Total MPs correlated negatively with thrombus weight in the DeltaCT group (p < 0.05). MP re-injections led to greater thrombus weight, while older MP reinjections tended to form larger thrombus than younger. Finally, TF bearing MPs showed a significant correlation to MP concentrations (R = 0.99). In conclusion, MPs appear to be an important element in venous thrombogenesis.

The cell adhesion molecule P-selectin mediates the interaction of activated platelets or endothelial cells with leukocytes. In arterial and venous thromboembolism (VTE) increased soluble P-selectin (sP-selectin) concentrations have been found, and associations of P-selectin genotypes with thrombotic disease have been proposed. We assessed the effect of four single nucleotide polymorphisms (SNPs) [one in the promoter region (c.-2123C>G) and three (S290N, c.1087G>A; D562N, c.1902G>A; T715P, c.2363A>C) in the coding region] and the calculated haplotypes in the P-selectin gene (SELP) on sP-selectin concentrations and VTE risk. The analysis was carried out in 116 high-risk patients with a history of objectively confirmed recurrent VTE and 129 age- and sex-matched healthy individuals. Haplotypes were generated using computer-assisted haplotype reconstruction with Phase 2.1. sP-selectin (microg/l) was measured by ELISA. Frequencies of all four individual SNPs were not statistically significantly different between patients and controls. Ten haplotypes were obtained for the control population, and nine for the patient group. The most frequent haplotype among controls was CGGA (major allele at all positions) (27.8%; frequency in patients 19.0%), which was used as reference for statistical analyses. Among patients GGAA was most frequent (23.3%; frequency in controls 17.5%). Haplotypes were significantly associated with sP-selectin concentrations in patients and in controls (p<0.001 and p=0.011). Compared to CGGA some but not all haplotypes conferred an increased risk for VTE with odds ratios (ORs) between 5.4 (95% CI: 2.5-12.2) for CAGA, 3.3 (1.2-9.2) for CGAC, and 2.4 (1.3-4.7) for GGAA. All ORs remained statistically significant after adjustment for the factor V Leiden mutation, located in close proximity to SELP on chromosome 1, as well as all other established risk factors for VTE. In conclusion, SELP haplotypes modulate plasma concentrations of sP-selectin and affect the risk of recurrent VTE.

Increased levels of soluble P-selectin (sP-selectin) have been shown in a number of different disorders, e.g. diabetes mellitus (DM) and cardiovascular disease (CVD). Several studies have attempted to demonstrate the association of the most intensively examined variant of P-selectin gene polymorphism (Thr715Pro) with sP-selectin levels in healthy subjects and in CVD, but contradictory data have been reported. To clarify the effect of Pro715 allele on the sP-selectin levels in type 2 DM, we analysed this polymorphism in diabetic patients and compared these data with sP-selectin levels. Type 2 DM patients (n = 119), 48 BMImatched non diabetic individuals - consisting mostly of overweight subjects - and 57 healthy volunteers were included in the study. TheThr715Pro polymorphism was analysed by PCR-RFLP, while sP-selectin levels were measured by ELISA. Significantly elevated sP-selectin levels were found in both DM and in overweight subjects compared to healthy controls. We confirmed previous reports that in healthy Pro715 allele carriers lower sPselectin levels could be measured; however, this difference was only significant in case of lean subjects. No significant difference was detected in sP-selectin level among DM and overweight individuals according to this genotype. However, significant difference was observed in sP-selectin levels in older DM patients compared to younger ones, but these levels were not accounted for by the Thr715Pro polymorphism. We suggest that in type 2 DM individuals, the significantly elevated sP-selectin levels are not due to the Thr715Pro P-selectin gene polymorphism.

Individuals with lupus anticoagulants (LA) are at risk for thromboembolism (TE). Chronic inflammation is an important characteristic in LA patients which may dispose for TE. Platelets play a key role in inflammation and TE. We therefore investigated gene polymorphisms as well as plasma levels of platelet receptors as predictors of TE in 107 LA patients. We compared 74 patients with a history of thromboembolic disease (TE+), 56 with venous thrombosis (VT), 12 with arterial thrombosis (AT), and six patients who had both, with 33 LA patients without previous thrombosis (TE-). The P-selectin Pro715 allele was slightly more frequent in VT (OR = 3.167,95% CI 0.955-10.503; p = 0.0594), but no patient with AT had this allele (OR = 0.099, 95 % CI 0.001-0.790; p = 0.0238) which therefore may protect from AT. Plasma levels of P-selectin, collected a median of 35 months (range 2-329 months) after the last thrombotic event, were higher in patients withVT (p = 0.0096) than in TE-, but not with AT (p = 0.4713). These high P-selectin levels were not explained by the P-selectin polymorphism. The CA repeat polymorphism in the 3'-noncoding region of CD154 was significantly associated with the development of AT (OR = 4.035,95 % CI 1.329-12.249; p = 0.0138). Plasma levels of CD154 were not significantly different among the subgroups. Thus, the Thr715Pro polymorphism of P-selectin and CA repeats of CD154 are differentiating between the risk for VT and AT. Further, soluble P-selectin is elevated in LA patients with previous VT, but its role to predict VT needs to be evaluated in prospective studies.

The aim was to investigate whether the Thr715Pro P-selectin polymorphism is associated with soluble P-selectin (sP-selectin) levels in individuals from different ethnic groups. Plasma sP-selectin and Thr715Pro (A/C) P-selectin gene polymorphism were measured in 237 white (106 females), 177 black African origin (92 females) and 201 South Asian (94 females) individuals living in England. All were free from coronary heart disease (CHD), stroke and other cardiovascular disease, diabetes, drug therapy for hypertension or high lipids, hormone replacement therapy or oral contraceptive pill. The Thr715Pro C allele was rare in blacks (0.8%) and intermediate in South Asians (3.0%) compared to whites (11.2%; p <0.001). sP-selectin levels were significantly lower in the individuals with the AC or CC compared to the AA genotype in both whites (-25% (95% C.I. -33.3 to -16.9); p <0.001) and South Asians (-25.2% (-40.5 to -6.1); p <0.012). There was insufficient power for this analysis in blacks. In conclusion, in whites and South Asians the C allele of the Thr715Pro P-selectin polymorphism is associated with lower sP-selectin levels. Lower levels of sP-selectin were not accounted for by this polymorphism in blacks, in whom the C allele was very rare.

P-selectin is rapidly translocated from platelet alpha-granules following activation. Intracellular cyclic AMP (cAMP) is a potent inhibitory pathway that results in global downregulation of platelet activation. While cAMP-dependent protein kinase (PKA) has long been considered as the main mediator of cAMP-dependent effects, no study has yet evaluated its effect on P-selectin expression in human platelets. Pretreatment of thrombin-stimulated platelets with forskolin resulted in a concentration- dependent inhibition of P-selectin expression that correlated with adenylyl cyclase activity. Inhibition of PKA with H-89 reversed cAMP-induced inhibition of P-selectin while cGMP-dependent protein kinase (PKG) inhibition with KT5823 significantly potentiated cAMP-dependent inhibition of P-selectin. Similar results were also observed in a platelet/neutrophil binding assay. In conclusion, cAMP-induced inhibition of P-selectin expression is, in large part, mediated through activation of PKA. PKG appears to be solicited for P-selectin expression when cAMP levels are elevated which suggest a cAMP/PKG-dependent pathway of platelet activation.

We investigated similarities in the signaling pathways elicited by the F2 isoprostane 8-iso-PGF2alpha and by low doses of U46619 to induce platelet activation. Both 0.01-0.1 micromol/L U46619 and 0.01-1 micromol/L 8-iso-PGF2alpha triggered shape change and filopodia extension, as well as adhesion to immobilized fibrinogen of washed platelets. At these doses the two platelet agonists failed to trigger secretion and aggregation, which were however induced by higher doses of U46619 (0.1-1 micromol/L). SB203580 (1-10 micromol/L), a specific inhibitor of the p38 mitogen activated protein (MAP) kinase blunted platelet shape change and adhesion induced by 0.05-1 micromol/L 8-iso-PGF2alpha and by 0.01 micromol/L U46619. These platelet responses were also inhibited by 20 micromol/L cytochalasin D, an inhibitor of actin polymerization, and 50 micromol/L piceatannol, an inhibitor of the Syk tyrosine kinases. Both 8-iso-PGF2alpha and U46619-induced p38 MAP kinase phosphorylation in suspended platelets and this was inhibited by piceatannol, indicating that Syk activation occurs upstream of p38 MAP kinase phosphorylation. These findings suggest that the signaling pathway triggered by both 8-iso-PGFalpha and low concentrations of U46619 to induce platelet adhesion and shape change implicates Syk, the p38 MAP kinase, and actin polymerization.

Genetically engineered mice bring animal studies to the molecular level in that they help establish the role of a particular molecule, or its portion, in a complex biological process. In recent years, several discoveries were made using the selectin-mutant mice. For example, it was shown that these molecules not only mediate leukocyte rolling, but also platelet rolling on the vessel wall. The functional significance of platelet rolling has yet to be uncovered. The process could be important for hemostasis leading to firm platelet adhesion at sites of denuded endothelium and/or in inflammation. After activation, platelets may help in leukocyte recruitment as shown by studies of lymphocyte homing to peripheral lymph nodes. Surprisingly, work with the P-selectin mutant mice has also revealed an anti-inflammatory aspect of platelet P-selectin. P-selectin binding to leukocytes promoted the transcellular production of an anti-inflammatory mediator limiting the extent of acute glomeluronephritis. In addition, soluble P-selectin was shown recently to be shed from both activated platelets and endothelium and there are strong indications that it too could have an attenuating effect on inflammatory disease progression. Another discovery made with the selectin-deficient mice is on the crucial role of P- and E-selectins in the homing of hematopoietic progenitor cells to the bone marrow. This observation could perhaps be further exploited by use of selectin inhibitors when liberating the progenitors from the marrow for transplant purposes. The use of selectin inhibitors could also be evaluated in two major disease processes where selectins were recently shown to play a role: cancer and atherosclerosis. Thus the selectin mutant mice have taught us a great deal about the role of selectins in normal physiology and in pathology. Further studies are needed to explore the regulation of shedding of the selectins and the function of soluble selectins in vivo. Exploring new territories of selectin-mediated interactions may provide a basis for developing new interventions and treatments for diseases in which the role of selectins has not yet been suspected.

Thrombopoietin (Mpl ligand), interleukin-6 (IL-6), and interleukin-11 (IL-11) differ in their effects on megakaryocyte maturation and development. In the present study, the impact of these thrombocytopoietic cytokines on biochemical and structural granule and membrane components was examined. Western blotting was performed on equivalent amounts of isolated megakaryocyte or platelet protein and the relative intensities of the enhanced chemiluminescent-visualized bands were quantitated by densitometry. Megakaryocyte growth and development factor (MGDF), a recombinant thrombopoietin-related molecule, significantly increased megakaryocyte fibronectin (72%), thrombospondin (55%), von Willebrand factor (28%) and p-selectin (CD62p) (37%) when compared to equivalent amounts of protein from saline-treated controls (p<0.01). Megakaryocyte fibrinogen was not increased. Ultrastructurally, there was a marked increase in ribosomes and rough endoplasmic reticulum even in mature-appearing megakaryocytes. Platelets from MGDF-treated mice showed small increases in fibronectin (8%), and CD62p (18%), but did not show increases in other measured alpha-granule proteins. Neither IL-6 nor IL-11 increased megakaryocyte or platelet alpha-granule proteins over levels observed in saline controls. IL-11 treated megakaryocytes, while also exhibiting an increase in ribosomes, were characterized by prominent cytoplasmic fragmentation. The study demonstrates the impact of Mpl ligand in increasing synthesized megakaryocyte alpha-granule proteins and of IL-11 in promoting megakaryocyte fragmentation.

Platelet membrane glycoproteins play a crucial role in ischemic complications after coronary stenting. Glycoprotein IIb-IIIa blockade reduces adverse clinical events after angioplasty but is associated with rare but profound thrombocytopenia that might increase hemorrhagic complications. Changes in platelet membrane glycoproteins of patients with angina who underwent coronary stenting and were treated with the GPIIb-IIIa antagonist abciximab (n=20) or with heparin (n=23) were studied. GPIb-IIIa receptor blockade and membrane glycoproteins were evaluated with immunological markers in venous blood samples taken before. 10, 24, 48, 72, and 96 h after initial treatment with either abciximab or heparin. Patients receiving abciximab therapy showed a rapid inhibition of binding of fluorochrome-conjugated mAb CD41 and c7E3 concomitant with a reduction in platelet aggregation which was restored in part in the days after termination of abciximab infusion. Induction of ligand-induced binding sites on GPIIb-IIIa was increased in patients receiving abciximab. The expression of ligand-induced binding sites correlated inversely with platelet count. No significant change in platelet membrane markers were found in the heparin group. In vitro studies showed that abciximab induces ligand-induced binding sites on isolated platelets and on nuclear cells bearing recombinant GPIIb-IIIa. Abciximab rapidly achieves GPIIb-IIIa receptor blockade after coronary stent placement that might be beneficial in high-risk settings to bridge the delayed action of ticlopidine. Significant alterations of platelet membrane glycoproteins during GPIIb-IIIa antagonism might contribute to development of acute profound thrombocytopenia.