BACKGROUND

Increased platelet activation has been described during treatment with various immunosuppressive agents and may contribute to the high cardiovascular mortality rate in renal transplant patients. Platelets are thought to propagate the inflammatory process of atherosclerosis by interaction with leukocytes.

METHODS

We tested an experimental therapy with clopidogrel in renal transplant patients treated with either tacrolimus (n = 20) or cyclosporine (INN, ciclosporin) (n = 19). All patients took low-dose steroids and had stable transplant function. Untreated healthy volunteers (n = 11) were included as the reference group. Degranulation (CD62P), glycoprotein IIb/IIIa receptor activation (PAC1), formation of platelet-leukocyte aggregates (monocyte-platelet-leukocyte aggregate, CD11b, mean fluorescence intensity), and platelet CD40 ligand (CD40L) expression (percent positive) were assessed by flow cytometry before therapy (visit 1) and after 4 weeks of clopidogrel (75 mg/d) intake (visit 2). To assess systemic anti-inflammatory effects, we measured levels of high-sensitivity C-reactive protein, soluble CD40L (sCD40L), monocyte chemoattractant protein 1, and matrix metalloproteinase 9 (MMP-9) by enzyme-linked immunosorbent assay.

RESULTS

At visit 1, cyclosporine-treated patients had significantly enhanced CD62P and PAC1 expression and platelet-leukocyte aggregate formation, as well as elevated sCD40L concentrations, compared with tacrolimus-treated patients (all P < .03). Clopidogrel intake led to a significant decrease in platelet-leukocyte aggregate formation in tacrolimus-treated patients (median, 237 [interquartile range, 177-510] to 194 [interquartile range, 159-275] mean fluorescence intensity; P < .035) and cyclosporine-treated patients (median, 450 [interquartile range, 362-782] to 254 [interquartile range, 211-458] mean fluorescence intensity; P < .035). CD40L expression was reduced in tacrolimus-treated patients (median, 34 [interquartile range, 28-41] to 21 [interquartile range, 12-26] mean fluorescence intensity; P < .002) and cyclosporine-treated patients (median, 33 [interquartile range, 30-37] to 26 [interquartile range, 19-26] mean fluorescence intensity; P < .02). In addition, CD62P, PAC1, and CD11b were significantly reduced in both groups at visit 2 (P < .02). MMP-9 decreased from 88 ng/mL (range, 49-135 ng/mL) to 57 ng/mL (range, 38-73 ng/mL) (P < .05) in tacrolimus-treated patients and from 79 ng/mL (range, 54-148 ng/mL) to 66 ng/mL (range, 41-97 ng/mL) (P < .01) in cyclosporine-treated patients. The sCD40L concentration decreased significantly only in cyclosporine-treated patients (P < .004). In contrast, high-sensitivity C-reactive protein and monocyte chemoattractant protein 1 were not affected.

CONCLUSIONS

The P2Y(12) receptor antagonist clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes. Because the synthesis of vascular disease markers and inflammatory products such as sCD40L and MMP-9 has been inhibited, anti-inflammatory properties of clopidogrel are likely to be a result of decreasing platelet activation.

CD18-deficient mice (CD18(-/-) mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E(-/-) mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18(-/-)CD62E(-/-), but not CD18(-/-)CD62P(-/-), mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10-15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E(-/-) mice that were reconstituted with CD18(-/-) bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-alpha-induced model of inflammation. Leukocyte adhesion efficiency in CD18(-/-)CD62E(-/-) mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18(-/-)CD62E(-/-) mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

Rapid tissue destruction in group A streptococcal (GAS) necrotizing fasciitis/myonecrosis often necessitates extensive debridement to ensure survival. The mechanisms responsible for this fulminant process remain unknown; we hypothesized that toxin-induced ischemia contributes to necrosis. In a rat model, Doppler flowmetry was used to measure local blood flow at the site of the intramuscular injection of exotoxins from an invasive M-type 1 GAS, which caused a rapid, dose-dependent decrease in perfusion that was irreversible at the highest toxin concentration tested. Videomicroscopic results revealed that blood flow was impeded by occlusive intravascular cellular aggregates. Flow-cytometric results confirmed that GAS toxins induced the coaggregation of platelets and neutrophils, that this activity was attributable to streptolysin O, and that platelet/neutrophil complex formation was largely mediated by platelet P-selectin (CD62P). Strategies that target platelet adherence molecules may prevent vascular occlusion, maintain tissue viability, and reduce the need for amputation in necrotizing GAS infections.

The aim of this study was to evaluate platelet activation in gastric cancer patients with regard to histopathological classification and the presence of distant metastases, by using platelet morphological parameters: MPV, L-PLT, MPC, as well as quantitative evaluation of surface receptor expression: CD41a, CD61, CD42b, CD62P, by flow cytometry at the resting state and after TRAP activation. In gastric cancer patients higher values of MPV and LP, as well as decreased MPC values were determined. Quantitative evaluation of surface antigen expression also revealed higher number of CD41a, CD61 and CD62P molecules, as compared with the platelets in the control group. Significant decrease of CD42b molecules' number after TRAP incubation, and the increased CD41a, CD61 and CD62P expression also point to the retained reactivation capacity of platelets. Good correlation between morphological parameters and the number of CD62P molecules indicates the usefulness of routine tests in evaluation of platelet activation.

At present, little is known about the clearance of platelet-derived microparticles (PMP) in human blood, as due to ethical considerations infusion experiments with labeled microparticles are delicate. Therefore, we investigated the kinetics of PMP, which are abundantly present in apheresis platelet concentrates (PC), following platelet transfusion in severe thrombocytopenic patients (n=11). PMP were double-stained with annexin V and cell-specific antibodies (anti-CD61, anti-CD63 or anti-CD62P, respectively) and detected by flow cytometry before and after transfusion of a single PC at fixed time intervals. Upon transfusion, the plasma levels of MP binding annexin V (2.5-fold), PMP (CD61+; 2.9-fold), and PMP from activated platelets (CD63+; 1.9-fold) or P-selectin (2.5-fold) increased immediately. The plasma levels of MP decreased with a half life of 5.8 hours (annexin V; 95% CI: 1.8?18.3) and 5.3 hours (CD61; 95% CI: 2.0?14.2). This is the first report in which the half life time of transfused PMP has been investigated in humans.

There is a growing body of evidence on the role of nitric oxide (NO) in human platelet physiology regulation. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO-), because it is formed by a number of diverse biochemical reactions. The aim of the present study was to comparatively analyze the effect of HNO and NO on several functional parameters of human platelets. For this purpose, sodium trioxodinitrate (Angeli's salt,AS) and sodium nitroprusside (SNP) were used as HNO and NO releasers, respectively. BothAS and SNP significantly inhibited platelet aggregation and ATP release induced by different agonists and adrenaline. AS or SNP did not modify the expression of platelet glycoproteins (Ib, IIb-IIIa, la-IIa, IV), whereas they substantially decreased the levels of CD62P, CD63 and of PAC-1 (a platelet activated glycoprotein IIb/IIIa epitope) after the stimulation with ADP. AS and SNP significantly increased cGMP accumulation in a 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ)-sensitive manner. However, while L-cysteine reduced the effect of AS, it increased the effect of SNP on this parameter. Accordingly, a differential effect of L-cysteine was observed on the antiaggregatory effect of both compounds. In summary, these results indicate that HNO is an effective inhibitor of human platelet aggregation.

The effects of dietary n-3 fatty acids (n-3FAs) on the frequency of pain episodes and ex vivo blood tests of thrombosis have been evaluated in patients with sickle cell disease (SCD) utilizing a double-blind, olive oil-controlled clinical trial. Dietary n-3FA therapy (0.1 g/kg/d) was provided as menhaden fish oil (0.25 g/kg/d) containing 12% eicosapentaenoic acid (EPA), and 18% docosahexaenoic acid (DHA). Within 1 month dietary n-3FAs exchanged with n-6FAs in plasma and erythrocyte membrane phospholipids (p <0.01 in all cases). Treatment with dietary n-3FAs for 1 year reduced the frequency of pain episodes requiring presentation to the hospital from 7.8 events during the preceding year to 3.8 events/year (p <0.01; n = 5). By contrast, subjects receiving control dietary olive oil (n = 5) experienced 7.1 pain events/year, compared to 7.6 during the previous year (p >0.4). The reduction in episodes in n-3FA-treated subjects was also significant when compared to control subjects (p <0.01). Dietary n-3FA therapy was not associated with hemorrhagic, gastrointestinal or other adverse effects. Compared to 10 asymptomatic African-American controls, sickle cell subjects demonstrated significantly increased pretreatment: 1) flow cytometric expression of platelet membrane P-selectin (CD62p; p <0.01) and annexin V binding sites (p = 0.02); 2) plasma levels of platelet-specific secretory proteins platelet factor 4 (PF4) and beta-thromboglobulin (betaTG) (p <0.01 in both cases); 3) plasma products of thrombin generation, prothrombin fragment 1.2 (F1.2) and thrombin:antithrombin (TAT) complex (p <0.01 in both cases); and 4) plasma levels of thrombolytic products, D-dimer and plasmin:antiplasmin (PAP) complex (p <0.01 in both cases). Treatment with dietary n-3FAs concurrently decreased plasma levels of F1.2, D-dimer, and PAP (p <0.05, compared to olive oil controls), implying that the reduction in pain events was related to n-3FA-dependent inhibition of thrombosis. We conclude that dietary n-3FAs reduce the frequency of pain episodes perhaps by reducing prothrombotic activity in sickle cell disease.

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.

Atherosclerotic cardiovascular mortality is increased in rheumatoid arthritis (RA) patients. We evaluated the association of inflammatory response with platelet, endothelial, coagulation activation parameters; and subclinical atherosclerosis in RA patients. We included 27 RA patients (21 female; six male) and 19 healthy subjects (14 female; five male). Disease activity score (DAS28) in RA patients was calculated; and patients were divided into two groups as active and inactive. Flow cytometry was used to determine platelet CD62P expression, platelet microparticles (PMP), platelet-monocyte (PMC) and platelet-neutrophil complexes (PNC). Plasma E-selectin, thrombin-antithrombin (TAT) complex, and serum sCD40L levels were determined by ELISA. The intima-media thickness (IMT) of carotid arteries was determined by B-mode ultrasonography. In RA patients, platelet CD62P expression (p < 0.001), PMC (p = 0.037) and sCD40L (p < 0.001) levels were increased when compared to the control group. PNC (p = 0.07) and TAT levels (p = 0.1) were non-significantly higher, and PMP level (p = 0.075) was nonsignificantly lower in RA patients. Soluble E-selectin level was significantly higher in the active RA group than in the inactive RA group (p = 0.009). There was no correlation between carotid IMT and activity markers, the evaluated parameters (p>> 0.05).The increase in markers of active platelets, CD62P and sCD40L, and PMC levels might be associated with the increased cardiovascular mortality in RA. Nevertheless, none of these parameters were associated with carotid IMT: this suggests that one cross-sectional value might not be a good marker for atherosclerosis

The pathogenesis of atherosclerosis, the leading cause of morbidity and mortality in industrial countries, is multifactorial. Atherogenesis, the development of atherosclerotic lesions, is initiated by a mechanical or functional injury of the endothelium. The function of the endothelium is influenced by multiple factors as a consequence of cell-cell interactions. Cell-cell communication between endothelial cells with platelets has only recently begun to receive systematic study. In recent years it has been established that platelet-endothelial interactions are involved at all stages of atherosclerotic disease. This article reviews the interactions between endothelial cells and platelets in the context of their role to initiate and accelerate atherothrombosis, as well as in acute thrombotic occlusion (e.g., at the site of atherosclerotic plaque rupture or subsequent to coronary angioplasty). From a mechanistic standpoint, platelets and endothelial cells communicate on multiple levels. Cross-talk may occur over a distance (paracrine signaling), via transient interactions (so-called give-and-go mechanism), or through receptor-mediated cell-cell adhesion. Platelets may release or transfer substances that influence endothelial cell function, and vice versa. Among many others, adhesion molecules, such as P-selectin (CD62P), are of special interest because of their role in modulating interactions between blood cells and the endothelium, and also because of the possible use of the soluble form as a plasma predictor of adverse cardiovascular events. In addition to dietary, cholesterol and lipid lowering, and other pharmaceutical approaches, antiplatelet therapy plays an important part in the treatment of atherosclerosis and its multifactorial clinical manifestations. Understanding the specific interactions between platelets and the endothelium may lead to the development of novel therapeutic strategies.

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.

Phosphatidylserine (PS) exposure serves as a procoagulant stimulus and a signal for phagocytic clearance of apoptotic cells. In order to measure PS exposure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leukocytes were identified by CD45 and side-scatter gating, and platelets by CD6 1 and side-scatter gating. The absolute number of annexin V molecules bound per cell was determined from an independent calibration procedure. Normal populations had the following levels of annexin V binding (in molecules per cell): lymphocytes, 0.53 x 10(3) neutrophils, 1.75 x 10(3) monocytes, 2.45 x 10(3) platelets, 0.14 x 10(3). These levels represent </= 0.1% of the values obtained after maximal stimulation of PS exposure with calcium ionophore, confirming that virtually all PS is intracellular in normal circulating leukocytes and platelets. Pretreatment of whole-blood samples with ammonium chloride to lyse erythrocytes caused a 9- to 300-fold increase in annexin V binding to leukocytes, indicating that analysis of unlysed whole-blood samples is essential to avoid artifactual increases in annexin V binding to leukocytes. Comparison of annexin V with two other markers of platelet activation, <em>CD62P</em> and the activation-dependent epitope of glycoprotein IIb/IIIa detected by the PAC I antibody, indicated that platelets from normal donors showed the least amount of activation with the annexin V marker. Whole-blood flow cytometry with annexin V can reliably measure the state of PS exposure in platelets and leukocytes, and the results confirm that these cell

It is reported that the incidence of thromboembolism is increased in ulcerative colitis (UC), and hypercoagulability persists even when patients are in remission. We evaluated the association of inflammatory response parameters with UC activity, and activation parameters of the platelets, endothelium, and the coagulation system in UC. Eighteen UC patients and 19 healthy subjects were included in the study. The patients' clinical features were recorded down; whole blood counts and acute phase parameters were evaluated. UC patients were divided into two as active (9 patients) and inactive (9 patients) according to combined clinical activity index (CAI) and endoscopic activity index (EAI) scores. In all subjects, platelet CD62P expression, platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), and platelet microparticles (PMP) were determined by flow cytometry. E-selectin, thrombin-antithrombin complex (TAT) levels in plasma, and sCD40L levels in serum were determined by ELISA. In both active and inactive UC patients, platelet CD62P expression, the percentages of PMC, and PNC were significantly higher than those in the control group (P< 0.01). PMP level was higher in the control group than in inactive UC patients (P = 0.001). sCD40L level was significantly higher in active UC group than in the control group (P < 0.01). EAI score correlated significantly with PMP (r = 0.5, P = 0.04) and sCD40L (r = 0.48, P = 0.044); CAI score had a negative correlation (r = -0.68, P = 0.002) with sE-selectin level. In addition to increased CD62P expression and sCD40L, increased formation of PMC and PNC suggests a role for platelet-leukocyte complex formation together with platelet activation in thromboembolic events observed in UC.

BACKGROUND

Transfusion of platelet concentrates (PCs) is the basic treatment for severe platelet disorders. PCs carry the risk of pathogen transmission, especially bacteria. Pathogen reduction (PR) by addition of photochemical reagents and irradiation with visible or ultraviolet (UV) light can significantly reduce this risk. We present a novel approach for PR in PCs employing UVC light alone.

METHODS

UVC PR was evaluated by bacteria and virus infectivity assays. PC quality was investigated by measuring pH, lactate, glucose, hypotonic shock response, platelet aggregation, CD62P expression, and annexin V binding as in vitro parameters. The impact of UVC PR on the platelet proteome was assessed by differential in-gel electrophoresis and compared with changes caused by UVB and gamma-irradiation, respectively.

RESULTS

Vigorous agitation of loosely placed PCs generated thin fluid layers that allow penetration of UVC light for inactivation of the six bacteria and six of the seven virus species tested. HIV-1 was only moderately inactivated. UVC light at the dose used (0.4 J/cm(2)) had a minor impact on in vitro parameters and on storage stability of treated PCs. Proteome analysis revealed a common set of 92 (out of 793) protein spots being affected by all three types of irradiation. Specific alterations were most pronounced for gamma-irradiation (45 spots), followed by UVB (11 spots) and UVC (2 spots).

CONCLUSIONS

UVC irradiation is a potential new method for pathogen reduction in PCs. The data obtained until now justify further development of this process.

Adhesion molecules on endothelial cells or platelets may regulate localization and activation of leukocytes at sites of tissue injury, infection, or thrombosis. In these studies, we found that human peripheral blood monocytes adhered specifically to immobilized P-selectin (CD62P), Chinese hamster ovary cells transfected with a cDNA for P-selectin, or endothelial cells stimulated to express P-selectin on the cell surface. P-selectin did not directly stimulate synthesis of the lipid autoacoid platelet-activating factor (PAF); however, incubation on immobilized P-selectin primed monocytes for increased synthesis of PAF in response to opsonized zymosan particles. P-selectin did not stimulate increased surface expression of integrin CD11b/CD18 and did not enhance binding of iC3b-coated erythrocytes, a CD11b/CD18-mediated functional response. P-selectin increased PAF production by monocytes incubated with unopsonized zymosan particles that stimulate this response by interaction with the beta-glucan receptor. Further, phagocytosis of unopsonized zymosan particles, another response triggered by the beta-glucan receptor, was increased following the adherence of monocytes to P-selectin. These data suggested that P-selectin primed monocytes for increased PAF synthesis through regulation of the beta-glucan receptor or regulation of signal transduction mechanisms that are linked to the receptor. P-selectin expressed on endothelial cells or platelets may serve both to localize monocytes at sites of vascular inflammation or thrombosis and to prime the cells for subsequent responses that augment inflammation.

OBJECTIVE

Platelet activation plays a crucial role in the pathophysiology of cerebral ischemia. The aim of this study was to investigate the contribution of platelet activation and leukocyte-platelet interactions to the disease.

METHODS

One hundred thirty-five patients with transient ischemic attack (TIA) or stroke were enrolled in this single-center study. They underwent cranial computer tomography within 24 hours of clinical onset and after 3 months, and systemic venous blood samples were drawn. Platelet activation (CD62P expression), leukocyte activation (L-selectin expression), and the appearance of platelet-specific antigens on leukocytes as an index of platelet-leukocyte aggregation were measured by flow cytometric techniques in the acute state and at 3-month follow-up.

RESULTS

Patients with a completed stroke or TIA had significantly increased circulating platelet-leukocyte aggregates, increased P-selectin expression on platelets, and decreased L-selectin expression in the acute state compared with the control group (healthy volunteers). No differences in regard to the tested activation markers could be detected between patients with stroke or TIA in the acute phase of the disease. However, platelet and leukocyte activations were normalized after 3 months in patients with TIA, whereas leukocyte activation (reduced L-selectin expression) remained in stroke patients.

CONCLUSIONS

In patients with TIA and completed stroke, platelet and leukocyte activation is substantially enhanced in the acute phase of the disease. The sustained leukocyte activation observed in stroke but not in TIA patients at 3-month follow up might play a pathophysiological role in the course of the disease.

BACKGROUND

There is increased platelet activation in many cardiovascular diseases. This observation may explain the presence of increased levels of platelet microparticles (PMP) in these diseases. However, whether or not levels of PMPs inter-relate with other markers of platelet activation, such as soluble P-selectin, or with disease severity, is unknown. We therefore hypothesized raised PMP levels in stable peripheral artery disease (PAD) intermittent claudication (IC), with an additional increase in severe PAD critical limb ischaemia (CLI). Furthermore, we tested the hypothesis that PMP levels are correlated with other markers of platelet activation, such as soluble P-selectin, membrane bound P-selectin (CD62P) and 63.

METHODS

Patients with PAD were recruited from the vascular outpatient and inpatient facilities at a teaching hospital. Age- and sex-matched controls were also recruited from healthy volunteers. Venous blood was obtained from 23 patients with severe disease (CLI), 36 with moderate disease (IC), and from 30 healthy controls. The percentage of platelets positive for CD62P and CD63, as well as the numbers of PMPs were defined by flow cytometry. Plasma soluble P selectin was measured by enzyme-linked immunosorbent assay (ELISA).

RESULTS

PMPs were increased relative to healthy controls in patients with IC, with a further increase in CLI (P<0.001). Soluble P selectin and CD62+ve platelets were raised in both patient groups, but there was no difference amongst the two patient groups. CD63+ve cells were raised only in CLI compared to healthy controls. In multivariate analysis, only PMP and soluble P selectin independently predicted disease severity, and the two markers correlated modestly (r=0.345, P<0.001).

CONCLUSIONS

Increased PMP and soluble P selectin are both related to the severity of symptomatic PAD. However, it is uncertain if this relationship is a cause or effect of atherosclerosis. This finding may have clinical implications as PMPs have the potential to influence the progression of atheroma as well as promote thrombosis.

The pathophysiologic features of stent-induced cellular responses of platelets and leukocytes have not been established. This study was designed to clinically investigate the activation of platelets and neutrophils after coronary stenting and to identify its effects on the long-term results of coronary stents. Forty-eight consecutive patients with left anterior descending coronary artery disease indicating coronary intervention were randomly assigned to either a balloon angioplasty group or a coronary stent group. Flow cytometric analysis demonstrated that the transcardiac gradient (the value of coronary sinus blood minus the value of peripheral blood) of platelet surface expression of CD62P (p < 0.001) and CD63 (p < 0.01) increased immediately after coronary stenting, but increased less significantly immediately after balloon angioplasty (CD62P, p < 0.01; CD63, p < 0.05). These increases were persistently observed after coronary stenting but transiently after balloon angioplasty alone during a 48-hour observation period after the procedures. The gradient for neutrophil surface expression of CD11b increased, and that of CD62 L decreased 48 hours after coronary stenting (CD11b, p < 0.001; CD62 L, p < 0.05), but these changes showed less significance 48 hours after balloon angioplasty alone (CD11b, p < 0.05; CD62 L, p = NS). The gradients 48 hours after the procedures for both CD62P (r = 0.39, p < 0.05) and CD11b (r = 0.44, p < 0.01) were independently correlated with the late loss in the stent group, whereas the correlation was seen only for CD11b (r = 0.38, p < 0.05) in the balloon angioplasty group. Both platelet and neutrophil activation was greater after coronary stenting than after balloon angioplasty. Cellular interactions between platelets and neutrophils may be related to the progression of neointimal proliferation leading to restenosis after coronary stent implantation.

BACKGROUND

Platelets play a key role in atherogenesis and thromboembolic complications in patients with type 2 diabetes.

RESULTS

We prospectively examined the relationship between systemic platelet activation and progression of carotid wall thickness within 1 year in 105 patients with type 2 diabetes. The intima-media thickness (IMT) of the common carotid artery was measured bilaterally at study entry and after 1 year. Platelet activation was assessed with the use of immunologic markers of platelet activation (CD62P, CD63, and CD40L) and flow cytometry. The prevalence for progression of atherosclerotic carotid disease in this population was 55.2%. We found that platelet degranulation (CD63 and CD40L) correlated with progression of IMT within 1 year (CD63: r=0.231, P=0.022; CD40L: r=0.230, P=0.029). Diabetic patients with progression of IMT had a significantly increased expression of CD63 compared with patients with stable carotid disease (mean intensity of immunofluorescence; median, interquartile range: 17.1 [12.4, 25.8] versus 11.9 [7.7, 19.8]; P=0.004). Multivariate logistic regression analysis revealed that degranulation of platelet CD63 is a predictor for progression of IMT independently of classical cardiovascular risk factors and hemoglobin A1c in diabetic patients (P=0.017).

CONCLUSIONS

Enhanced systemic platelet degranulation is associated with progression of carotid artery disease in type 2 diabetes.

BACKGROUND

Microparticles released from platelets may play a role in the normal hemostatic response to vascular injury, because they exhibit prothrombinase activity. Microparticles are generated by high shear stress and may be formed in diseased small arteries and arterioles in various clinical settings. However, the surface composition of high shear-induced platelet microparticles is unknown. It was recently shown that some cytokines modulate platelet activation. However, no reports are available concerning the effect of cytokines on high shear-induced platelet aggregation (SIPA) microparticle generation.

METHODS

Measurement of SIPA was performed with a cone-plate viscometer. The conformational characteristics of high shear (108 dynes/cm(2))-induced platelet microparticles were analyzed by flow cytometry and confocal laser scanning microscopy. Effects of cytokines for high SIPA microparticle generation were also analyzed using flow cytometry.

RESULTS

The overall pattern of monoclonal antibody binding in high shear-induced microparticles was almost the same as that in activated platelets under high shear stress. Microparticles exhibited markedly increased Annexin V binding. In fluorescent confocal images, small and fine regions of fluorescence (microparticles) were recognized separate from platelet fluorescence. Thrombopoietin not only induced platelet activation, as demonstrated by CD62P expression, but also increased the number of microparticles. Erythropoietin and interleukin-6 enhanced only microparticle generation.

CONCLUSIONS

These results suggest that microparticles possessing procoagulant activity are released by platelet activation when levels of certain cytokines increase under high shear stress in various clinical settings.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

BACKGROUND

Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated.

METHODS

Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation.

RESULTS

As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression.

CONCLUSIONS

Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.

Injection of monosodium urate (MSU) crystals, the etiological cause of gouty arthritis, into murine peritoneal cavities produced an intense recruitment of polymorphonuclear leukocytes (PMN). After 3 mg MSU crystal injection, cell influx was maximal (approximately 10 x 10[6] cells per mouse) at 6 hr postinjection and sustained up to the 24 hr time-point. In mice depleted of mast cells by administration of compound 48/80 72 hr before challenge with MSU crystals a lower PMN influx was measured (58% reduction). The occurrence of endogenous mast cell activation, in the MSU response, was validated by the observation that MSU challenge reduced by more than 90% the number of intact mast cells recovered in the peritoneal washes. Pretreatment of mice with a histamine H1 antagonist (tripolidine; 0.5 mg/kg) or a platelet-activating factor receptor antagonist (WEB2086; 10 mg/kg) significantly reduced by 50 to 60% the number of PMN recovered from the peritoneal cavities. The molecular determinants of this process of leukocyte recruitment were also investigated. Treatment of mice with an anti-CD62P or anti-CD62E monoclonal antibody (mAb; 100 microg i.v.) produced a distinct inhibition of PMN recruitment measured at 6 hr, whereas only a combined administration of both monoclonal antibodies was effective in reducing by 60% the influx of PMN caused by the MSU crystals within 24 hr. In conclusion, these data highlight a role for endogenous mast cells and for endothelial-derived selectins in MSU crystal-induced PMN recruitment into the peritoneal cavity, and may be useful to dissect molecular mechanism(s) which may be operating in gouty arthritis.

Thromboembolism and bleeding remain significant complications of ventricular assist device (VAD) support. Increasing the amount of biocompatibility data collected during preclinical studies can provide additional criteria to evaluate device refinements, while design changes may be implemented before entering clinical use. Twenty bovines were implanted with the EVAHEART centrifugal VAD for durations from 30 to 196 days. Titanium alloy pumps were coated with either diamond-like carbon or 2-methoxyethyloylphosphoryl choline (MPC). Activated platelets and platelet microaggregates were quantified by flow cytometry, including two new assays to quantify bovine platelets expressing CD62P and CD63. Temporally, all assays were low preoperatively, then significantly increased following VAD implantation, before declining to a lower, but still elevated level over 2-3 weeks. MPC-coated VADs produced significantly fewer activated platelets after implant trauma effects diminished. Three animals receiving no postoperative anticoagulation had similar amounts of circulating activated platelets and platelet microaggregates as animals receiving warfarin anticoagulation. Two new methods to quantify bovine activated platelets using antibodies to CD62P and CD63 were characterized and applied. These measures, along with previously described assays, were able to differentiate between two biocompatible coatings and assess effects of anticoagulation regimen in VAD preclinical testing.