Meera Mody

Flow cytometric analysis of platelet function in stored platelet concentrates

Platelet activation occurs during the collection, processing and storage of platelet concentrates. The effect of the platelet activation on the functional state of stored platelets remains however undefined. We employed flow cytometric analysis to evaluate the extent of platelet activation and the physiological response to thrombin stimulation of platelets stored for up to five days under routine blood bank conditions. Platelet surface expression of the activation markers CD62 and CD63 was examined, along with modulation of platelet membrane glycoproteins (GP) Ib and IIbIIIa. Platelet dense granule content was determined using a mepacrine uptake assay and the extent of platelet microparticle generation was quantified. Thirteen random-donor platelet concentrates prepared under routine conditions by a platelet-rich-plasma protocol were examined. Platelets were found to be activated following preparation on day 1. Although a gradual increase was seen with increasing storage time, this was not statistically significant for CD62 or CD63 expression, GPIIbIIIa or GPIb modulation or dense granule release; the generation of platelet microparticles did, however, increase with increasing storage time. The characteristic increase in surface expression of CD62, CD63 and GPIIbIIIa and decrease in GPIb and dense granule content in response to thrombin stimulation was observed with all concentrates, but these measures of platelet functional reserve showed decreasing platelet function with increasing storage time. The results indicate that platelets are activated by day 1, likely as a consequence of manipulation during collection and processing, but are not further progressively activated with increasing storage time; they do, however, become relatively hypofunctional with increasing storage.

Flow cytometric analysis of platelets from children with the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure.

The pathophysiology of platelet dysfunction in the Wiskott‐Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet‐associated IgG and reduced platelet‐dense granule content. Levels of reticulated ‘young’ platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar‐sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin‐ and ADP‐induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F‐actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.

Comparison of platelet immunity in patients with SLE and with ITP

Idiopathic thrombocytopenic purpura (ITP) is characterized by the development of a specific anti-platelet autoantibody immune response mediating the development of thrombocytopenia. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of a wide variety of autoantibodies. In 15-20% of SLE cases, patients develop thrombocytopenia which appears to be autoimmune in nature (SLE-TP). To better understand the pathogenesis of the thrombocytopenia associated with SLE, we investigated the overlapping platelet and cellular immune features between SLE and ITP. Thirty-one patients with SLE, eight with SLE-TP, and 17 with ITP, were studied and compared to 60 healthy controls. We evaluated platelet-associated IgG, platelet microparticles, reticulated platelets, platelet HLA-DR expression, in vivo cytokine levels, lymphocyte proliferation, and the T lymphocyte anti-platelet immune response in these patients. Patients with SLE-TP and those with ITP had increased platelet-associated IgG, an increased percentage of platelet microparticles, a higher percentage of reticulated platelets and larger platelets, suggesting antibody-mediated platelet destruction and increased platelet production. More than 50% of patients with ITP had increased HLA-DR on their platelet surface whereas subjects with SLE-TP did not. Analysis of serum cytokines demonstrated increased levels of IL-10, IL-15 and TNF-alpha in patients with SLE, but in those with ITP, only increased levels of IL-15 were seen, no increases in any of these cytokines were observed in patients with in SLE-TP. The ability of lymphocytes to proliferate in response to phorbol myristate acetate (PMA) stimulation was increased in SLE-TP, but was normal in both SLE and ITP. Lymphocytes from subjects with ITP displayed an increased ability to proliferate on exposure to platelets, in contrast, those with SLE-TP did not. While the number of subjects evaluated with SLE-TP was small, these data reveal a number of differences in the immunopathogenesis between SLE-TP and ITP.

Characterization of HIV-1-specific antibodies and HIV-1-crossreactive antibodies to platelets in HIV-1-infected haemophiliac patients

Sera from HIV‐1‐infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate, HIV‐p24 and HIV‐gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid‐sensitive and alkaline‐sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV‐lysate columns; 96% (25/26) of the acid‐eluted antibodies were HIV‐specific but 48% (12/25) of the alkaline‐eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline‐eluted HIV‐p24 antibodies, 40% (8/20) reacted specifically with HIV‐p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline‐eluted HIV‐gpl20 antibodies (n=17), 88% (15/17) were HIV gpl20‐specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti‐p24 antibodies recognized three bands with approximate molecular weights of 72 000 to 95 000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti‐HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline‐sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV‐infected haemophiliacs.

Platelet-surface glycoproteins in healthy and preeclamptic mothers and their newborn infants

Preeclampsia, a common complication of pregnancy, contributes significantly to maternal and fetal morbidity and mortality. It may lead to both quantitative and qualitative defects of maternal and neonatal platelets. In this prospective study, flow cytometry has been used to study expression of platelet-surface glycoproteins (GPs) on maternal and neonatal platelets of both healthy and preeclamptic subjects. We studied 15 preeclamptic women, 20-44 y of age, and their newborns (median gestational age, 32 wk; range, 26-38) and seven healthy women (aged 26-41 y) and their healthy newborns(median gestational age, 38 wk; range, 38-42). Compared with their healthy and preeclamptic mothers, resting platelets from neonates expressed significantly less CD41 and CD9. Thrombin activation resulted in significant increases in platelet-surface expression of CD62P, CD63, CD41, CD9, and CD36 in neonates and their healthy mothers. Compared with neonates of healthy mothers, platelets from neonates of preeclamptic mothers expressed lower levels of CD62P, CD63, CD9, and CD36 on activated platelets. These findings suggest that preeclampsia influences the expression of platelet-surface GPs on neonatal and maternal platelets, which may affect platelet function, leading to an additional risk for bleeding in thrombocytopenic neonates of mothers with preeclampsia.

The Relationship of von Willebrand Factor Binding to Activated Platelets from Healthy Neonates and Adults

von Willebrand Factor (VWF) is important in platelet adhesion and shear-dependent platelet activation. We performed flow cytometric analyses of VWF binding to and activation of platelets from healthy neonates, children, and adults. Platelets from cord blood (n = 38; gestational age: 36–42 wk; birth weight: 2.4–5.1 kg), neonatal venous blood (n = 19; d 2–3 of life), children (n = 15; age: 1.5–16.3 y), and adults (n = 22; age: 18–55 y) were studied. Binding of VWF was assessed using an antihuman VWF polyclonal antibody and a FITC-conjugated secondary antibody. Platelet activation was determined by the expression of CD62P, CD63, CD41, CD42b, activated GPIIb/IIIa (PAC-1), procoagulant surface (as reflected by annexin V binding), and microparticle formation. Although the mean percentage of VWF-positive platelets was not significantly higher in unstimulated platelets from 2- to 3-d-old neonates, their platelets were more activated than those from adults, and there was a positive correlation of VWF binding with platelet activation (CD62P:r = 0.74, p < 0.001; annexin V:r = 0.46, p < 0.05). In adults, after in vitro activation of platelets with thrombin and ADP, VWF binding to platelets increased and correlated significantly with CD62P expression (r = 0.71, p < 0.001). VWF binding to unstimulated neonatal platelets was, however, higher than that to in vitro–stimulated platelets from adults at the same level of expression of platelet activation markers. Further studies are required to assess the mechanism and significance of VWF binding to activated platelets in the neonatal period.

A rabbit model for monitoring in vivo viability of human platelet concentrates using flow cytometry

BACKGROUND : Viability in vivo of novel platelet components cannot be readily determined in human transfusions. Elaboration of valid animal models may be useful for this purpose.

Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen‐activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine–threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal‐related kinases (ERKs), c‐Jun amino‐terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF‐induced human platelet activation. It is not known whether VWF‐dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF‐dependent platelet activation, we functionally inhibited ERK‐dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK‐1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF‐ or thrombin‐induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF‐ or thrombin‐dependent platelet activation.

p38 MAPK is activated but not necessary in porcine von Willebrand factor-dependent platelet activation

We have investigated the role of p38 mitogen‐activated protein kinase (MAPK) in von Willebrand factor (VWF)‐dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF‐dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post‐addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S‐transferase‐MAPK activated protein kinase‐2. To determine if p38 MAPK was necessary for porcine VWF‐induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF‐induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580‐inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function

Summary.  The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF‐dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF‐dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF‐dependent platelet activation was compared to their ability to inhibit thrombin‐dependent activation. The results demonstrate that VWF‐dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin‐sensitive phosphatidylinositide 3‐kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin‐dependent platelet activation. In addition, thrombin‐dependent but not VWF‐dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin‐ and VWF‐dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin‐dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF‐dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF‐dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.