anti-CD62P Antibody Research Articles

C1-inhibitor influence on platelet activation by thrombin receptors agonists.

IntroductionProtease activated receptors 1 (PAR1) and 4 (PAR4) agonists are used to study platelet activation. Data on platelet activation are extrapolated across experimental settings. C1-inhibitor (C1INH) is a protease inhibitor present in plasma but not in isolated platelet suspensions. Here we show that C1INH affects platelet activation through PAR1 and PAR4 agonists.MethodsPlatelets were isolated from healthy donor whole blood and then labeled with anti-CD62P and PAC1 antibodies. The platelet suspensions were exposed to PAR1 agonists SFLLRN, TFLLR and TFLLRN; PAR4 agonists AYPGKF and GYPGQV; ADP and thrombin. Flow-cytometric measurements were performed in 5, 10 and 15 min after activation.Results0.25 mg/ml C1INH addition made platelets to faster expose CD62P and glycoprotein IIb/IIIa complex after activation with PAR1 agonists. Conversely, C1INH addition led to inhibition of platelet activation with PAR4 agonists and thrombin. Activation with ADP was not affected by C1INH.ConclusionsOur results suggest that C1INH can modify platelet activation in the presence of synthetic PAR agonists used in platelet research. These observations may be relevant to the development of new methods to assess platelet function.

Platelets function as an acute viral reservoir during HIV-1 infection by harboring virus and T-cell complex formation

AbstractPlatelets were recently found to harbor infectious HIV virions in infected individuals who are on antiretroviral treatment with poor CD4+ T-cell recovery. In this study, we screened platelets from recently infected individuals, before and after antiretroviral therapy, for the presence of virus and examined platelet activation, as well as CD4+ T-cell recovery. This was followed by in vitro studies assessing platelet–CD4+ T-cell complex formation as a contributing factor to viral transmission. HIV+ platelets were detected in 10 of 10 acutely infected individuals with no prior history of antiretroviral therapy. The percentage of HIV+ platelets dropped significantly after 3 months of antiretroviral therapy in all of the study participants. These individuals also demonstrated significant recovery of CD4+ T cells. Interestingly, the percentage of HIV+ platelets correlated positively with viral load but not with CD4+ T-cell count. Furthermore, we found that platelet activation with soluble CD40L or thrombin receptor activator peptide 6 (TRAP6) increased platelet-virus interactions in vitro. TRAP6-mediated interactions were reduced by platelet antagonists, aspirin, and R406. We demonstrated that platelets transmit the virus to CD4+ T cells, and this transinfection was abolished by inhibiting platelet–T-cell complex formation via exposure to an anti-CD62P antibody. Additionally, treatment with TRAP6 significantly increased the transinfection, which was also inhibited by aspirin and R206. These results reveal that platelets have the potential to promote HIV viral spread during the acute stage of infection, by harboring infectious virus transmitting infection to susceptible CD4+ T cells through complex formation.

Modulation of Thrombin-Induced Platelet Activation By Defibrotide

Defibrotide is a mixture of single-stranded phosphodiester oligonucleotides 9-80 bases in length derived from mammalian tissue and it is the only medication that is FDA approved for the prevention and treatment of post-transplant veno-occlusive disease, also known as sinusoidal obstructive syndrome (VOD/SOS). The mechanism of action of defibrotide is not well understood. A better understanding of the molecular mechanism by which defibrotide prevents and reverses the microvascular occlusion seen in VOD/SOS will be critical in the development of novel therapeutics for VOD/SOS and other related disorders. Our central hypothesis is that single stranded phosphodiester oligonucleotide mixtures such as defibrotide alter thrombin-induced platelet and endothelial cell activation primarily via altered signaling through protease activated receptors (PARs). To determine the effect of defibrotide on thrombin-induced platelet activation we performed flow cytometry on washed platelets from humans and mice. Washed platelets were incubated with agonist (1 nM thrombin, 100 μM synthetic PAR1 agonist TRAP6, or 100 μM synthetic PAR4 agonist PAR4AP) in the presence or absence of 1 mg/mL of defibrotide. Fluorescent antibodies against P-selectin (anti-CD62P antibody) and against the high affinity conformation of the αIIβ3 receptor (PAC1 antibody for human samples and JON/A antibody for murine samples) were added to assess for inhibition of platelet activation by defibrotide. Fluorescent anti-PAR1 antibodies SPAN12 (which spans the canonical thrombin cleavage site) and WEDE15 (which binds at the hirudin-like domain) were also added to separate samples to assess whether defibrotide inhibits cleavage of the PAR1 N-terminal domain by thrombin. After twenty minutes of incubation at room temperature the samples were analyzed by flow cytometry. We calculated the geometric mean of the fluorescence intensity (MFI) for three replicate samples and data are shown as the fold change compared to the washed platelet control without addition of agonist or defibrotide. For thrombin-stimulated human platelets, both anti-CD62P binding (MFI fold over control 5.7 vs. 1.2) and PAC1 antibody binding (MFI fold over control 7.1 vs. 1.7) are significantly reduced in the presence of defibrotide (See Figure 1a). For thrombin-stimulated murine platelets there is a small decrease in anti-CD62P antibody binding (MFI fold over control 8.4 vs. 6.6), but no significant change in JON/A antibody binding (See Figure 1b). Binding of both anti-CD62P and PAC1 antibodies to human platelets stimulated with synthetic PAR agonists was unaffected by the presence of defibrotide (See Figure 1c). There is no significant difference in SPAN12 or WEDE15 binding in thrombin-stimulated platelets in the absence or presence of defibrotide. However, defibrotide alone does appear to interfere with WEDE15 binding in unstimulated platelets (See Figure 1d). In conclusion, we showed that defibrotide inhibits thrombin-induced human platelet activation at low concentrations of thrombin, as shown by a decrease in surface platelet activation markers measured by flow cytometry. This effect is much less pronounced under the same conditions if mouse platelets are used, supporting the hypothesis that defibrotide inhibits platelet activation by alteration of PAR1 receptor signaling, since PAR1 receptors are not present on mouse platelets. Defibrotide does not interfere with TRAP6-induced platelet activation, evidence against interference with the intracellular portion of the signaling pathway, since TRAP6 is a synthetic PAR1 agonist that binds directly into the ligand-binding pocket of the receptor, eliminating the need for cleavage of the PAR1 extracellular domain. The decrease in WEDE15 antibody binding to washed platelets seen with defibrotide alone suggests that the mechanism may involve alterations in the hirudin-like domain of the PAR1 receptor which is crucial for thrombin binding and cleavage site specificity. Ongoing studies including investigating the effect of defibrotide on peptide mimics of the extracellular N-terminal domain of the PAR1 receptor and the effect of defibrotide on the activation of endothelial cells will likely shed light on the exact mechanism of platelet response modulation by defibrotide and may provide new targets for treatment of the microvascular occlusion that characterizes VOD/SOS. Disclosures No relevant conflicts of interest to declare.

A new hydrophilic polysulfone hemodialysis membrane can prevent platelet-neutrophil interactions and successive neutrophil activation.

Purpose:Microaggregates have often been observed during hemodialysis and are clearly associated with complications of hemodialysis therapy. In this study, we aimed to clarify the effects of two polysulfone membranes, with different abilities to activate blood cells, on the formation of these microaggregates; we also investigated their molecular mechanisms.Methods:Human whole blood was circulated through a mini-module dialyzer using the membranes in vitro; platelet–neutrophil complexes in blood were determined by flow cytometry. Isolated human neutrophils were incubated with the membranes in plasma, in the presence or absence of platelets, followed by flow cytometric analysis of intracellular reactive oxygen species and cell-surface activated CD11b on neutrophils.Results:CX-U, a conventional polysulfone membrane with remarkable cell activation, induced the formation of platelet–neutrophil complexes; however, NV-U, a new hydrophilic polysulfone membrane with slight or no cell activation, did not cause complex formation. Moreover, CX-U-induced reactive oxygen species production and the increase in activated CD11b expression on neutrophils were enhanced by platelets. On the other hand, NV-U hardly affected neutrophil activation, regardless of whether platelets were present or not. The enhancement of CX-U-induced neutrophil activations by platelets was greatly inhibited by anti-CD62P antibody.Conclusion:The ability of polysulfone membranes to activate blood cells is closely related to platelet–neutrophil interaction. Therefore, a biocompatible membrane, like NV-U, can be expected to prevent microaggregate formation during hemodialysis and avoid subsequent cell activation.

Differential Sensitivity of Various Markers of Platelet Activation with Adenosine Diphosphate

A number of techniques have been available to assess platelet activation, but their relative sensitivity is unknown and their usage is variable and not based on any rational criteria. Here, we compared the ability of several techniques based on morphological and biochemical markers to detect the first signs of ADP-induced platelet activation. Scanning electron microscopy of platelets was performed in parallel with flow cytometry to quantify the surface expression of P-selectin (marked by labeled anti-CD62P antibodies), active αIIbβ3-intergrin (assessed by the binding of labeled fibrinogen) and phosphatidylserine (assessed by the binding of labeled Annexin V). When expressed as a fraction of activated platelets, shape changes were the most sensitive to a low ADP concentration compared to the biochemical markers in the following order of sensitivity: morphological changes>fibrinogen binding capacity>P-selectin expression> phosphatidylserine exposure. These results suggest the greater sensitivity of platelet microscopy and the importance of its combination with flow cytometry used to detect surface expression of the molecular markers of platelet activation.

The impact of hyperglycemia and hyperlipidemia on plasma P-selectin and platelet markers after ischemic stroke

IntroductionPlatelet activation plays a key role in the pathogenesis of ischemic cerebrovascular diseases. Thus, it is very important to identify novel pharmacological targets for platelet inhibition to improve ischemic stroke treatment. The aim of the study was to assess the relationship between metabolic disorders and platelet activity markers in patients with acute ischemic stroke.Material and methodsNinety-four patients with acute ischemic stroke were divided into four groups with: normolipidemia and normoglycemia (NL/NG), n = 25; normolipidemia and hyperglycemia (NL/HG), n = 21; hyperlipidemia and normoglycemia (HL/NG), n = 27; hyperlipidemia and hyperglycemia (NL/NG), n = 21. Twenty-one healthy subjects served as controls. We assessed the CD62P expression on resting and thrombin-activated blood platelets using the flow cytometer and anti-CD61 and anti-CD62P monoclonal antibodies. CD61-positive microparticles were defined as platelet-derived microparticles. The level of sP-selectin in serum was measured by the ELISA method.ResultsWe observed a significant influence of hyperlipidemia and hyperglycemia on sP-selectin concentration. A strong correlation between higher sP-selectin concentration and enhanced LDL (p = 0.001), total cholesterol (p = 0.02), HbA1c level (p < 0.001) was noticed. The level of sP-selectin and PDMPs (p < 0.001) were significantly higher in all groups of stroke patients compared with the controls. CD62P expression on resting and thrombin activated platelets were significantly lower in groups of patients with stroke.ConclusionsHyperlipidemia and hyperglycemia exert an equal stimulatory effect on tested platelet markers but with no synergistic action in stroke patients with both of the metabolic comorbidities. sP-selectin concentration in stroke patients best reflects the impact of hyperglycemia and hyperlipidemia on vascular lesions and platelet activation.

The sweeter aspects of platelet activation: A lectin-based assay reveals agonist-specific glycosylation patterns

BackgroundThe diversity of platelet functions implies multiple activation states arising in response to different stimuli. Distinguishing between these states has been challenging. MethodsWe used fluorescently labelled carbohydrate binding proteins lectins to investigate agonist-induced changes in platelet surface glycosylation. ResultsEach of the seven agonists we used caused a unique set of changes in platelet surface glycosylation, eliciting a unique functional state. Some of these changes could be correlated with the expression of granule-specific markers CD62P and CD63, but lectins proved much more sensitive to differences between agonists than antibodies against those markers. This sensitivity appears to arise from the relation between the surface glycosylation changes and the signalling pathways through which various agonists act. In this context it is interesting that the effects of calcium ionophore were significantly different from those of other agonists. We also found that that P-selectin (CD62P) contains haptens for lectins VFA and PTII, because these lectins compete with the anti-CD62P antibody binding and vice a versa. ConclusionsWe report for the first time that changes in platelet surface glycosylation are agonist-specific and can be distinguished using lectin-binding assays. Lectin fingerprinting represents a new research and diagnostic tool for studying platelet activation. General significanceThe observation of agonist-specific platelet surface glycosylation changes is interesting in the context of the diversity of platelet function, because surface glycans mediate contact interactions between platelets and other cells and serve as binding sites for some of the agonists (galectins).

The basis examination of leukocyte–platelet aggregates with CD45 gating as a novel platelet activation marker

Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation. Whole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5 μm thrombin receptor-activated peptide (TRAP) or 2 μg/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population. We analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.

Development of an improved assay system for activated platelet counts and evaluation by aspirin monitoring

Platelets represent a linkage among inflammation, thrombosis, and atherogenesis, and enhanced platelet activation is regarded as a risk for thrombotic disorders. The level of P-selectin expressed (CD62P) on the platelet surface is a useful marker of activated platelets (aPLT). Although CD62P has been measured briefly by flow cytometry using an anti-CD62P antibody, the assay remains imprecise and we tried to establish stable conditions for its measurement. The levels of aPLT are increased significantly by many factors, such as meals, sampling and keeping conditions, centrifugation, and the timing of fixation. For optimal results, sampling should be performed quickly in a K(2)-ethylenediaminetetraacetic acid (EDTA) containing a sample tube, and whole blood should be fixed with 666 mmol/L formaldehyde plus 167 mmol/L glyoxal for 5 min. After washing with phosphate buffered saline (PBS), the fixed platelets were reacted with anti-CD62P antibody for 20 min and measured by flow-cytometric detection for aPLT. The coefficient of variation of our aPLT assay was 10.4%. We also examined basic experiments to test the clinical application of our aPLT assay by monitoring aspirin therapy. The levels of aPLT after the administration of aspirin for 3 days were significantly lower than those in the group that did not receive aspirin. These results suggest that the aPLT assay is an effective analytical procedure for measuring platelet reactivity.

The Influence of Hyperlipidemia on Platelet Activity Markers in Patients after Ischemic Stroke

Background: To investigate the relationship between hyperlipidemia and platelet activation markers – platelet and soluble P-selectin (sP-selectin), and platelet-derived microparticles (PDMPs) – in patients after ischemic stroke. Methods: 41 patients after ischemic stroke (>3 months) confirmed by CT were divided into 2 groups: with hyperlipidemia (HL, n = 21) and normolipidemia (NL, n = 20). Twenty healthy subjects served as controls. CD62P-positive platelets and PDMPs in whole blood were analyzed by the use of a flow cytometer and anti-CD61 and anti-CD62P monoclonal antibodies. Platelets were activated by thrombin (0.08 units). The level of sP-selectin in serum was measured by ELISA. Results: We observed a significantly higher CD62P expression and percentage of CD62P-positive resting and thrombin-activated platelets in the HL as compared to the NL group. The sP-selectin concentration was also significantly higher in HL than NL subjects (p < 0.05). Moreover, we observed a significantly higher percentage of PDMPs in patients after stroke (NL: p < 0.05; HL: p = 0.005) in comparison with the control group. Conclusions: Patients after stroke present symptoms of platelet hyperreactivity. HL in the patients may be a risk factor for vascular events due to the increase in platelet activation.

Platelet Interaction with Bone Marrow Derived CD34+ Stem Cells: Potential Role of Chemoattractant Chemerin

Chemerin is a 16 kDa chemoattractant circulating in blood in an inactive form (prochemerin). Its chemotactic activity is released following proteolytic cleavage of its labile carboxyl-terminal amino acids by proteases of the coagulation, fibrinolytic and inflammatory cascades. Chemokine-like receptor 1 (CMKLR1) is a chemerin receptor expressed on plasmacytoid dendritic cells, natural killer cells and tissue macrophages. We identified the expression of CMKLR1 on mouse CD34 positive (CD34+) bone marrow-derived stem cells (BMSC) using anti-mouse CMKLR1 antibody by FACS. Fluorescein-labeled CD34+/CMKLR1+ BMSC adhered to immobilized chemerin dose-dependently. The EC50 of BMSC adhesion to human and mouse chemerin is 2.5 and 5 ug/mL respectively. BMSCs also adhered to SDF-1 (stromal cell-derived factor-1) with EC50 of 0.3 ug/mL, but did not bind to immobilized fibronectin. We have previously reported that platelets express chemerin and release it upon activation. CMKLR1 is also expressed on platelet surface as demonstrated by FACS. Immobilized chemerin could induce platelet adhesion and CD62P (p-selectin) expression as detected by fluorescein-labeled anti-CD62P antibody. BMSC formed complexes with both resting and activated platelets as demonstrated by fluorescein-labeled washed platelets incubated with unlabeled mouse CD34+/CMKLR1+ BMSCs using FACS analysis. Both anti-chemerin and anti-CD62P antibodies could partially block BMSCs binding to immobilized platelets at 50% and 35% respectively. In conclusion, chemerin directly mediates platelets interaction with BMSCs. Chemerin also facilitates platelet/BMSC complex formation by inducing platelet p-selectin (CD62P) expression. Platelet-derived chemerin, similar to SDF-1, may be involved in tissue regeneration by inducing progenitor cells mobilization and differentiation.

SB 497115-GR, a Low Molecular Weight TPOR Agonist, Does Not Induce Platelet Activation or Enhance Agonist-Induced Platelet Aggregation in Vitro.

SB-497115-GR is a low molecular weight, orally active molecule that requires Tpo receptor (TpoR) expession for activity and activates JAK/STAT signalling in platelets. The ability of recombinant Tpo to enhance agonist-induced platelet aggregation is well-documented, thus this study was undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01–10 uM) to affect platelet activation. Platelets were obtained from healthy human volunteers, following written informed consent, by standard venipuncture technique. Citrated whole blood was used directly in platelet activation and P-selectin assays within 15 minutes. Platelet aggregation was assessed by light transmittance aggregometry. Aggregation was induced by a submaximal concentration of adenosine diphosphate (ADP) (1.0 or 1.5 uM) in experiments examining synergy with rhTpo (150ng/mL) or SB-497115 (0.01–10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM), collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20 uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115, potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect on normal ADP, Collagen or TRAP-induced aggregation was seen with either SB-497115-GR or rhTpo. In experiments investigating the expression of platelet P-selectin, an early marker of platelet activation, platelets labeled with anti-CD62P antibody were analyzed by flow cytometry. Data was expressed as percent of platelets positive for CD62P within the defined platelet gate. Treatment with SB-497115 (0.1 – 10 uM) demonstrated no induction of platelet CD62P expression above background levels. In contrast, rhTpo produced a modest but significant and reproducible increase in the percent of platelets positive for CD62P. This study demonstrates that, in contrast to rhTpo, the orally active small molecular weight agonist of the Tpo receptor, SB-497115-GR, does not mimic the ability of rhTpo to enhance ADP-induced aggregation of platelets. SB-497115-GR also did not directly induce P-selection expression on human platelets. Finally, SB-497115 has no antagonistic effect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus, SB-497115 is differentiated from rhTpo with respect to platelet activation.

Cilostazol inhibits platelet–leukocyte interaction by suppression of platelet activation

The influence of three anti-platelet drugs, cilostazol, aspirin, and tirofiban, was investigated on platelet–leukocyte interaction by flow cytometry. When platelets and leukocytes were pre-incubated with anti-platelet drugs and stimulated by thrombin or collagen, cilostazol was found to inhibit platelet adhesion to monocytes and polymorphonuclear cells (PMNs). Similar effects were observed with anti-CD62P antibody, while aspirin and tirofiban did not appear to interfere with interaction between platelets and leukocytes. In the platelets pre-incubated with anti-platelet drugs, cilostazol significantly reduced CD62P expression and GPIIb/IIIa activation on platelet surface stimulated by thrombin or collagen. Aspirin inhibited CD62P expression and GPIIb/IIIa activation induced by collagen, but not thrombin. Tirofiban significantly blocked GPIIb/IIIa activation induced with both, and weakly inhibited CD62P expression induced by collagen. When added after stimulation of platelets, cilostazol again significantly inhibited CD62P expression and GPIIb/IIIa activation, although to a lesser extent than in the pre-incubation study. Aspirin hardly inhibited CD62P expression or GPIIb/IIIa activation, while tirofiban strongly blocked GPIIb/IIIa activation induced by thrombin or collagen, but had little effects on CD62P expression. In conclusion, our results suggest that cilostazol inhibits platelet–leukocyte interaction by reducing CD62P expression on the platelet surface.

Effects of high platelet concentration in collecting and freezing dry platelets concentrates

Objective. To evaluate, in vitro, the effects of collecting and cryopreserving fresh dry platelet concentrates (PCs). Material and methods. Standard and dry PCs were collected in the same apheresis procedure. PCs were evaluated by mean platelet volume (MPV), pH, glucose and LDH levels. Activation was examined by flow cytometry using anti-CD41, anti-CD42 and anti-CD62p monoclonal antibodies and annexin binding assay. Platelet function was assessed by aggregation using ADP, collagen and arachidonic acid as agonists. Dry PCs were compared to standard PCs and to cryopreserved dry PCs. We also compared the use of ThromboSol (TS) to 5% DMSO as cryoprotectives. Results. Dry PCs presented a significantly reduced pH and glucose ( p<0.001), increased LDH levels and CD62p expression ( p<0.001) and diminished aggregation response to ADP ( p<0.001). Platelet cryopreservation was associated with platelet lysis, activation and loss of function. Dry PCs cryopreserved with TS were associated with statistically higher LDH levels ( p<0.001) and a higher percentage of annexin binding ( p=0.005), in addition to a lower number of CD42 positive platelets ( p=0.01). Conclusion. Dry PCs should be rapidly frozen after collection to avoid a fall in pH and platelet activation. 5% DMSO performed better than TS to cryopreserve dry PCs.

Platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils

Activated platelets are known to adhere to both blood monocytes and neutrophils, and this adhesion is mainly mediated by the surface exposure of the platelet granule protein CD62P. Platelets as well as platelet-derived microvesicles (PMV) have also been shown to contain and to transfer tissue factor (TF), the most important initiator of intravascular thrombin and fibrin formation, to monocytes. However, the role of neutrophils for gathering platelet-derived TF is controversial. Here we studied the interaction of PMV with monocytes and neutrophils using a whole blood system. Platelet-rich plasma (PRP) obtained from citrated human blood was incubated with collagen (5 μg/ml, 15 min) and the platelets were removed by centrifugation (5 min at 5000 × g). After incubating the PMV-containing plasma for further 30 min with a sediment of red and white bloods cells that had been obtained after PRP preparation, monocytes and neutrophils were analysed by flow cytometry for the surface exposure of the platelet-specific antigen CD42a and TF. Compared to a control with non-activated PRP, there was a significant increase in the number of both CD42a-positive monocytes and neutrophils. In contrast, there was no change in the number of TF-positive neutrophils, but a more than 2-fold increase in the number of TF-positive monocytes. The changes in CD42a on monocytes and neutrophils as well as the changes in TF on monocytes could be significantly reduced by an anti-CD62P antibody or by removal of PMV from the plasma samples. The data indicate that the transfer of TF to monocytes is not simply an CD62P-mediated adhesion of platelets or PMV to monocytes, but may involve other not yet identified mechanisms.

Antibodies against human cell receptors, CD36, CD41a, and CD62P crossreact with porcine platelets

A limitation in platelet study has been the availability of platelet function-specific membrane receptor antibodies for use in the various animal species that are currently used in the study of hemostasis and other phenomena. Platelets were isolated from human and porcine blood. Resting and activated platelets were incubated with antibodies against the human cell surface receptors CD36 (clone CB38), CD41a (clone HIP8), CD62P (clone AK4), and CD63 (clone H5C6). Antibody titration and ligand blocking studies also were performed. Binding of anti-CD41a and anti-CD62P were similar for human and porcine platelets in percentage of platelets labeled and in number of receptors per cell. Binding of anti-CD36 was similar between species, with fewer receptors present in porcine cells. Anti-CD63 and anti-CD107a did not bind specifically to porcine platelets. The anti-CD36, anti-CD41a, and anti-CD62P antibodies studied crossreact with porcine platelets and will be useful in the investigation of platelet function in porcine models.

Activated Platelets Enhance Microparticle Formation and Platelet-Leukocyte Interaction in Severe Trauma and Sepsis

Activated platelets have been recently reported to produce platelet microparticles and to enhance platelet-leukocyte interaction. The precise role of platelets in systemic inflammatory response syndrome (SIRS) has not been clarified. The objective of this study was to evaluate microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. Twenty-six patients with severe SIRS (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 12 healthy volunteers were studied. The severe SIRS was caused by trauma in 12 patients and sepsis in 14. Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding, and platelet-polymorphonuclear leukocyte (PMNL) binding were measured by flow cytometry in the presence or absence of ionomycin, N-formyl-methionyl-leucyl-phenylalanine, or anti-CD62p monoclonal antibody. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood were also measured. Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding with or without ionomycin, and platelet-PMNL binding with ionomycin significantly increased in patients with severe SIRS in comparison with values in normal volunteers. The increased platelet-leukocyte binding in severe SIRS patients was markedly inhibited by P-selectin blockade and was not enhanced by N-formyl-methionyl-leucyl-phenylalanine. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood also increased in these patients. Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. Enhanced platelet-leukocyte interaction is dependent on P-selectin expression and may be involved in the systemic inflammatory response after severe inflammatory insult.

Platelet Activation Induced by Combined Effects of Anticardiolipin and Lupus Anticoagulant IgG Antibodies in Patients with Systemic Lupus Erythematosus

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p<0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+ x LA+ plasma only, but was significantly augmented by aCL+ x LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+ x LA-, aCL- x LA+ and aCL- x LA- plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+ x LA+ plasma (aCL+ x LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+ x LA--IgG and aCL- x LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.

Quantitative assessment of platelets, platelet microparticles, and platelet aggregates with flow cytometry

With fluorescent beads it has become possible to determine absolute numbers of cells in a given sample instead of relative percentages on a standard flow cytometer. This study assesses the ability to count platelets, microparticles, and aggregates with a flow cytometer. Whole blood was stimulated with 0.1 U thrombin per milliliter. Platelet and microparticle counts decreased, while the number of aggregates increased. Unactivated whole blood was diluted with buffer and showed a corresponding decrease in the concentration of platelets, microparticles, and aggregates. The platelet count on the flow cytometer was always in good correlation with counts on an automated blood analyzer. Only the cytometer, and not the automated analyzer, was able to detect and count microparticles and aggregates. In highly diluted samples of unactivated whole blood there was a spurious relative increase in CD62p-positive platelets because of a surplus of anti-CD62p antibodies and a relative increase in microparticles. Flow cytometry is a valuable method for counting platelets, aggregates, and microparticles in unstimulated and activated blood samples. If the platelet count changes and drops to less than 50% of the count for which the amount of antibody and the cytometer settings have initially been adjusted, care has to be taken to avoid misinterpretation.