Increased Responsiveness of Stored Platelets after Short-Term Refrigeration.

Introduction: Refrigeration of platelets is considered to provide advantages in therapy of acute hemorrhage due to increased platelet responsiveness. The alleviation of inhibitory signaling caused by cold temperature (CT) has been identified as an important mechanism contributing to enhanced platelet reactivity, detectable in freshly prepared platelets within 1 h of cold storage. The aim of this study was to confirm the effects of short-term refrigeration in platelets from apheresis-derived platelet concentrates (APC). Methods: APC were stored under standardized conditions for 1 day or for 2 days at room temperature and then refrigerated for 1 h, followed by sampling of platelets for analysis. Platelet reactivity was measured by aggregation studies using threshold concentrations of different agonists and by detection of fibrinogen binding using flow cytometry. The exploration of inhibitory signaling comprised the detection of VASP phosphorylation using flow cytometry or Western blot and the measurement of cyclic nucleotide levels. Results: Aggregation responses induced with ADP, collagen, or thrombin receptor-activating peptide-6 (TRAP-6) were increased in APC after cold storage for 1 h, associated with elevated TRAP-6-induced fibrinogen binding. VASP phosphorylation levels were decreased after cold exposition, detectable in 1-day- and 2-day-stored APC with flow cytometry, and in 2-day-stored APC with Western blot technique. Induced cGMP levels were lower after storage at CT in APC on day 1 and on day 2, whereas cAMP levels were reduced on 2-day-stored APC. Conclusion: Short-term refrigeration for 1 h is sufficient to induce an attenuation of inhibitory signaling, accompanied with increased aggregation responses in APC stored for up to 2 days. The "on demand" refrigeration of PC may be a reasonable approach for the preparation of platelets with enhanced responsiveness to treat patients with hemorrhage more effectively, which should be further addressed in consecutive studies.

Composition of plasma in apheresis-derived platelet concentrates under cold storage.

BACKGROUND: Compared to room temperature (RT, 22-24°C) storage, refrigeration of platelet concentrates (PC) may provide advantages due to lower risks of bacterial growth and increased responsiveness of platelets. However, storage at cold temperature (CT, 2-6°C) may also strongly influence the plasmatic composition of PC. This study analysed the content of plasma in apheresis-derived platelet concentrates (APC). MATERIALS AND METHODS: APC were stored under blood bank conditions at CT or RT. On days 0 and 6, samples were drawn for analysis. Coagulation parameters comprised global coagulation assays, single factors or inhibitors. The distribution pattern of von Willebrand multimers was investigated by immunoblotting. Thrombin generation was assessed with a fluorescence assay. Immunological and clinical chemistry parameters were determined on automated analysers. RESULTS: After storage at CT, coagulation factors V, VII, IX or protein S activity are partially reduced, but less compromised than under RT. There was a large reduction in Factor VIII levels and this was similar at both temperatures. In contrast to RT, von Willebrand Factor (vWF) activity was remarkably decreased at CT, and this was accompanied by the shift from high molecular to low molecular weight multimers. Thrombin generation showed improved preservation at CT. Other plasma proteins like immunoglobulins were stable at both conditions. DISCUSSION: Refrigeration mediates a bivalent effect on plasmatic coagulation in APC. At CT, the partial reduction of labile coagulation factors is less emphasised. However, CT does not prevent Factor VIII depletion, but induces an additional loss of vWF activity by multimer cleavage. Preserved thrombin generation may indicate a higher hemostatic capacity for cold storage.

The effect of short-term refrigeration on platelet responsiveness.

Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we could show that cold-induced attenuation of inhibitory signaling is an important mechanism promoting platelet reactivity. For developing strategies in blood banking, it is required to elucidate the time-dependent onset of facilitated platelet activation. Thus, freshly prepared platelet-rich-plasma (PRP) was stored for 1 and 2 h at CT (2-6 °C) or at RT (20-24 °C), followed by subsequent comparative analysis. Compared to RT, basal and induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation levels were decreased under CT within 1 h by approximately 20%, determined by Western blot analysis and flow cytometry. Concomitantly, ADP- and collagen-induced threshold aggregation values were enhanced by up to 30-40%. Furthermore, platelet-covered areas on collagen-coated slides and aggregate formation under flow conditions were increased after storage at CT, in addition to induced activation markers. In conclusion, a time period of 1-2 h for refrigeration is sufficient to induce an attenuation of inhibitory signaling, accompanied with an enhancement of platelet responsiveness. Short-term refrigeration may be considered as a rational approach to obtain PC with higher functional reactivity for the treatment of hemorrhage.

Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands.

Background Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets. Materials and Methods Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H 2 DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet-neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging. Results Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet-neutrophil contacts. Conclusion Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils.

Profile of the single-use, multiple-pass protein A adsorber column in immunoadsorption.

BACKGROUND AND OBJECTIVES: Immunoadsorptions (IA) are used to remove autoantibodies from the plasma in autoimmune disorders. In this study, we evaluated the effects of a single-use, recombinant staphylococcal protein A-based immunoadsorber on blood composition of the patient. MATERIALS AND METHODS: In a cohort of patients with myasthenia gravis or stiff-person syndrome, essential parameters of blood cell count, coagulation, clinical chemistry or plasma proteins and immunoglobulins (Ig) were measured before and after IA (n = 11). RESULTS: In average, IA reduced the levels of total IgG, IgG1, IgG2 and IgG4 by approximately 60%, the acetylcholine receptor autoantibody levels by more than 70%. IgG3, IgA or IgM were diminished to a lower extent. In contrast to fibrinogen or other coagulation factors, the column markedly removed vitamin K-dependent coagulation factors II, VII, IX and X by approximately 40%-70%. Accordingly, international normalized ratio and activated partial thromboplastin time were increased after IA by 59.1% and 32.7%, respectively. Coagulation tests almost returned to baseline values within 24 h. Blood cell count, electrolytes, total protein or albumin were not essentially affected. No clinical events occurred. CONCLUSION: The single-use, multiple-pass protein A adsorber column is highly efficient to remove IgG1, IgG2 and IgG4 or specific acetylcholine receptor autoantibodies from the plasma. Coagulation parameters should be monitored, since the column has the capacity to largely reduce vitamin K-dependent factors.

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib.

INTRODUCTION: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition. MATERIALS AND METHODS: Citrate-anticoagulated whole blood was stored with 5 nM and 1 µM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining. RESULTS: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces. CONCLUSIONS: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.

The involvement of toll-like receptors 2 and 4 in human platelet signalling pathways.

In addition to haemostasis, platelets play an essential role in mechanisms of inflammation and in immunological reactions. Platelets express various toll-like receptors (TLR) on their surface, among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. This study compared TLR2- and TLR4-dependent platelet signalling and their effect on platelet function. Platelet-rich-plasma and washed platelets were prepared from peripheral blood samples of healthy donors. Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) were used for stimulation of TLR2 and TLR4. Intracellular signalling pathways were investigated by Western blot. TLR2- and TLR4-mediated specific transcription factor DNA binding activity was measured by the nuclear factor kappa B (NFκB) transcription factor assay kit. Platelet adhesion and glycoprotein Ib function were assessed by immunofluorescence staining and analysis of ristocetin-induced agglutination. Both, Pam3CSK4 and LPS were able to induce NFκB-mediated and classical activating platelet signalling with a higher stimulatory capacity of TLR2. In addition, TLR2 and TLR4 activation led to a similar activation of inhibitory pathways. In contrast to TLR2, stimulation of TLR4 resulted in decreased Akt/protein kinase B phosphorylation conditioned by enhanced protein phosphatase 2A activity. TLR4-mediated signalling induced platelet adhesion and facilitated ristocetin-induced platelet agglutination. In conclusion, Pam3CSK4 directly induces aggregation via classical activation cascades, whereas LPS enhances platelet adhesion and glycoprotein receptor Ib-dependent platelet agglutination.

The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness.

Introduction Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies.

The role of proteasome activity for activating and inhibitory signalling in human platelets.

Platelets express key proteins of the proteasome system, but its functional role in the regulation of platelet integrity, however, is not fully understood yet. Therefore, this study evaluated activating and inhibitory platelet signalling pathways using the potent and selective proteasome inhibitor bortezomib. In washed platelets, the effect of bortezomib on viability and on aggregation was assessed. In addition, fibrinogen binding and CD62P expression were determined. The influence on activating and inhibitory signalling was detected by phosphorylation levels of essential messenger molecules. Platelet viability was maintained after incubation with 0.01 µM to 1 µM bortezomib, but tampered with 100 µM bortezomib. Agonist-induced aggregation was only reduced under 100 µM bortezomib and with weak induction by 10 µM adenosine diphosphate. Similarly, phosphorylated kinase levels of the activating signalling pathways were not affected by 0.01 µM to 1 µM bortezomib. In contrast, proteasome inhibition resulted in the reduction of inhibitor-induced vasodilator-stimulated phosphoprotein phosphorylation, accompanied with the partial decrease of induced inhibition of fibrinogen binding and CD62P expression. In conclusion, platelet activation and aggregation are not dependent on proteasome activity. Instead, inhibitory signalling is partially attenuated under proteasome inhibition. Supramaximal inhibitory concentrations of bortezomib (above 1 µM) lead to heterogeneous effects on activating or inhibitory systems, probably caused by decreasing platelet viability.

The Role of Human Platelet Preparation for Toll-Like Receptors 2 and 4 Related Platelet Responsiveness.

Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs). Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli ) as ligands. The capacity of chemokine release was determined by immunoassays. Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L. Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine.

The role of agonist-induced activation and inhibition for the regulation of purinergic receptor expression in human platelets.

INTRODUCTION: Adenosine diphosphate (ADP) as physiological activator of human platelets mediates its effects via three purinergic receptors: P2Y1, P2Y12 and P2X1. The inhibition of P2Y12 is used pharmacologically to suppress aggregation underlining the physiological significance of this receptor. Since the regulation of purinergic receptor expression has not thoroughly been investigated yet, this study analyzed the content of purinergic receptors on the platelet surface membrane upon activation and inhibition. MATERIALS AND METHODS: The surface expression of purinergic receptors was measured by flow cytometry using two different polyclonal antibodies as basal values and after incubation with thrombin receptor activating peptide (TRAP-6) or with inhibitors DEA/NO, MAHMA/NO or Prostaglandin E1 (PGE1). Western blot analysis was used to confirm inhibitory effects. RESULTS: Both investigated antibodies revealed a significant increase of purinergic receptor expression upon TRAP-6 stimulation. The NO donors, DEA/NO and MAHMA/NO, did not influence basal or TRAP-6 stimulated values. PGE1 did not affect basal receptor expression, but diminished TRAP-6 stimulated purinergic receptor expression in a dose-dependent manner. CONCLUSIONS: In summary, TRAP-6 induced platelet activation leads to an elevation of purinergic receptor expression. In contrast to other surface ligands, this effect is not suppressed by cGMP-mediated inhibition, but almost completely abrogated by enhanced cAMP-mediated signaling as induced by PGE1.

The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions.

BACKGROUND: Platelets are important for effective hemostasis and considered to be involved in pathophysiological processes, e.g. in cardiovascular diseases. Platelets provided for research or for therapeutic use are frequently separated from citrated whole blood (WB) stored for different periods of time. Although functionally intact platelets are required, the stability of platelet integrity, e.g. adenosine diphosphate (ADP) mediated responsiveness, has never been thoroughly investigated in citrated WB under ex vivo conditions. OBJECTIVES: Platelet integrity was evaluated at different time points in citrated WB units, collected from healthy donors and stored for 5 days at ambient temperature. The analysis included the measurement of activation markers, of induced light transmission aggregometry and of purinergic receptor expression or function. Inhibitory pathways were explored by determination of basal vasodilator-stimulated phosphoprotein (VASP)-phosphorylation, intracellular cyclic nucleotide levels and the content of phosphodiesterase 5A. Fresh peripheral blood (PB) samples served as controls. RESULTS: On day 5 of storage, thrombin receptor activating peptide-6 (TRAP-6) stimulated CD62P expression and fibrinogen binding were comparable to PB samples. ADP induced aggregation continuously decreased during storage. Purinergic receptor expression remained unchanged, whereas the P2Y1 activity progressively declined in contrast to preserved P2Y12 and P2X1 function. Inhibitory pathways were unaffected except for a slight elevation of VASP phosphorylation at Ser239 on day 5. CONCLUSION: After 5 days of storage in citrated WB, platelet responsiveness to TRAP-6 is sufficiently maintained. However, ADP-mediated platelet integrity is more sensitive to deterioration, especially after storage for more than 2 days. Decreasing ADP-induced aggregation is particularly caused by the impairment of the purinergic receptor P2Y1 activity. These characteristics should be considered in the use of platelets from stored citrated WB for experimental or therapeutic issues.

Evaluation of dose-dependent effects of the proteasome inhibitor bortezomib in human platelets.

Platelets express key proteins of the proteasome system and contain protein ubiquitination pathways. The functional role of the proteasome system in platelets, however, is still subject of studies. In addition to its role as anticancer drug, the potent and selective proteasome inhibitor bortezomib can be used for experimental proteasome research. Since it is mandatory to know exact dose-effect relationships, we intended to evaluate dose-dependent specific bortezomib effects on basal and on agonist-induced proteasome activitiy, on levels of poly-ubiquitinated proteins and on platelet aggregation. In washed platelets, unstimulated or stimulated with different agonists and pre-incubated with various bortezomib concentrations, the proteasome activity was determined by a fluorometric assay. The levels of poly-ubiquitinated proteins were assessed by an immunoassay kit. Platelet aggregation was measured by light transmission aggregometry in platelet-rich-plasma. Platelet agonists stimulate both, the proteasome activity and the accumulation of poly-ubiquitinated proteins in platelets. Bortezomib inhibits the basal and the agonist induced proteasome activity and increased the content of poly-ubiquitinated proteins in a concentration dependent manner. Bortezomib concentrations in the nM-range causing complete blockade of platelet proteasome activity do not affect agonist induced platelet aggregation, indicating that the level of platelet proteasome activity is not directly linked with the induction of platelet aggregation. Bortezomib in the µM-range may tamper platelet aggregation, possibly due to unspecific and toxic effects.

Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets.

BACKGROUND: Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms-potentially interfering with experimental results-have not been thoroughly studied. OBJECTIVES: Since ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation. RESULTS: In contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets. CONCLUSION: In conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function.

Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates.

BACKGROUND: The storage of platelets affects platelet integrity and functionality, a process named platelet storage lesion (PSL). Reduced adenosine diphosphate (ADP)-induced platelet aggregation is a typical manifestation of PSL. However, the role of ADP receptors in this context has not been evaluated yet. The aim of this study was, therefore, to investigate surface expression and function of the purinergic receptors P2Y1, P2Y12 and P2X1 in stored platelet concentrates. MATERIAL AND METHODS: Platelets were obtained from venous whole blood and from apheresis-derived platelet concentrates stored for 0, 2 and 5 days. Purinergic receptor expression was measured by flow cytometry and western blot analysis. Receptor function was determined by calcium-induced fluorescence (P2Y1 and P2X1) or by flow cytometric measurement of the platelet reactivity index (P2Y12). RESULTS: The basal surface expression and total content of purinergic receptors remained unchanged throughout storage. After an initial reduction during apheresis, P2X1-mediated calcium flux was maintained, whereas the P2Y1-mediated increase of calcium flux gradually decreased during the course of storage. In contrast, the platelet reactivity index was comparable in freshly obtained and stored platelets. DISCUSSION: The function of the P2Y12 receptor is maintained during storage of apheresis-derived platelet concentrates. However, the impairment of P2X1 and especially of P2Y1 receptor function indicated by decreased receptor-mediated calcium flux is an important mechanism contributing to reduced ADP responsiveness of stored platelets.

Specific inhibitory effects of the NO donor MAHMA/NONOate on human platelets.

Nitric oxide (NO) is a physiological inhibitor of platelet function and has vaso-dilating effects. Therefore, synthesized NO releasing agents are used e.g. in cardiovascular medicine. The aim of this study was to characterise specific effects of the short living agent MAHMA/NONOate, a NO donor of the diazeniumdiolate class, on human platelets. Whole blood was obtained from healthy volunteers. In washed human platelets, the MAHMA/NONOate induced phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and cyclic nucleotide production were studied by Western Blot and by enzyme immunoassay kits. Agonist induced aggregation was measured in platelet rich plasma. Paired Student׳s t-test was used for statistical analysis. MAHMA/NONOate significantly stimulated platelet VASP phosphorylation in a concentration dependent manner and increased intracellular cGMP, but not cAMP levels, transiently. ODQ, a specific inhibitor of the soluble guanylyl cyclase, completely prevented VASP phosphorylation induced by low MAHMA/NONOate concentrations (5nM-15nM). The effects of higher concentrations (30-200nM) were only partially inhibited by ODQ. MAHMA/NONOate reduced platelet aggregation induced by low doses of agonists (2µM ADP, 0.5µg/mL collagen, 5µM TRAP-6) in a concentration dependent manner. MAHMA/NONOate leads to a rapid and transient activation of platelet inhibitory systems, accompanied by decreased platelet aggregation induced by low dose agonists. At low MAHMA/NONOate concentrations, the effects are cGMP dependent and at higher concentrations additionally cGMP independent. The substance could be of interest for clinical situations requiring transient and subtotal inhibition of platelet function.

Increasing susceptibility of nitric oxide-mediated inhibitory platelet signaling during storage of apheresis-derived platelet concentrates.

BACKGROUND: Storage of platelets (PLTs) affects PLT integrity and functionality, a process named the PLT storage lesion. Normal PLT function essentially depends on the balanced interaction of activating and inhibitory signaling pathways. As there are poor data on the alterations of inhibitory signaling during storage of PLT concentrates, this study investigates the modulation capability of the cyclic nucleotide-mediated inhibitory pathways by use of the nitric oxide donor diethylamine diazenium diolate (DEA/NO). STUDY DESIGN AND METHODS: PLTs were obtained from whole blood (WB) and from apheresis-derived PLT concentrates (APCs) stored for 0, 2, and 5 days. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation, cyclic nucleotide concentrations, fibrinogen binding, and agonist-induced aggregation were measured without or after stimulation with DEA/NO. RESULTS: DEA/NO-induced VASP phosphorylation was significantly higher in PLTs from APCs on Days 2 and 5 compared to WB, conditioned by a stronger increase of cyclic guanosine monophosphate (cGMP), but not cyclic adenosine monophosphate (cAMP), in stored PLTs. A quantity of 5 nmol/L DEA/NO neither influenced thrombin receptor activator peptide 6 and collagen-induced aggregation nor fibrinogen binding in freshly collected PLTs, whereas it significantly inhibited both in stored PLTs. CONCLUSION: Stored PLTs showed an impairment of intracellular cGMP regulation, resulting in exceeding inhibition of agonist-induced aggregation and fibrinogen binding in the course of storage. The observed effects could be an important mechanism contributing to the storage lesion with reduced activating potential of PLTs.

Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis-derived platelet concentrates.

BACKGROUND: Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to agonists and impaired PLT activation, secretion, and aggregation. To understand the role of inhibitory signaling pathways in the mechanism of PSL, the basal state of the cyclic nucleotide (CN)-dependent signaling systems in stored PLTs was investigated. STUDY DESIGN AND METHODS: Whole blood samples (WB) and apheresis-derived PLT concentrates (APCs) were obtained from healthy volunteers. Washed PLTs were prepared from WB on Day 0 and from APCs on Days 0, 2, and 5. The basal phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and phosphodiesterase 5A (PDE5A) levels were quantified by Western blot. CN and PDE5A activity were measured by enzyme-linked immunoassay kits. Fibrinogen binding and aggregation were measured in PLT-rich plasma of WB or APC samples. Unpaired t test was used for statistical analysis. RESULTS: Basal VASP phosphorylation levels were comparable in WB and APCs on Day 0. VASP phosphorylation increased significantly during storage of APCs, more pronounced at Ser(239) than at Ser(157) . Similarly, intracellular cGMP, but not cAMP, concentration continuously increased in stored PLTs, whereas PDE5A levels and activity significantly decreased accompanied by diminished thrombin receptor activator peptide 6-induced fibrinogen binding and aggregation. CONCLUSION: Storage of APCs leads to intracellular cGMP accumulation that could be caused by degradation of PDE5A. Enhanced cGMP level supports subsequent cGMP-dependent protein kinase-mediated increase of VASP phosphorylation resulting in reduced fibrinogen binding and aggregation.

The oligopeptide DT-2 is a specific PKG I inhibitor only in vitro, not in living cells.

BACKGROUND AND PURPOSE: cGMP is involved in the regulation of many cellular processes including cardiac and smooth muscle contractility, aldosterone synthesis and inhibition of platelet activation. Intracellular effects cGMP are mediated by cGMP-dependent PKs, cGMP-regulated PDEs and cGMP-gated ion channels. PKG inhibitors are widely used to discriminate PKG-specific effects. They can be divided into cyclic nucleotide-binding site inhibitors such as Rp-phosphorothioate analogues (Rp-cGMPS), ATP-binding site inhibitors such as KT5823, and substrate binding site inhibitors represented by the recently described DT-oligopeptides. As it has been shown that Rp-cGMPS and KT5823 have numerous non-specific effects, we analysed the pharmacological properties of the oligopeptide (D)-DT-2 described as a highly specific, membrane-permeable, PKG inhibitor. EXPERIMENTAL APPROACH: Specificity and potency of (D)-DT-2 to inhibit PKG activity was evaluated using biochemical assays in vitro and by substrate phosphorylation analysis in various cell types including human platelets, rat mesangial cells and rat neonatal cardiomyocytes. KEY RESULTS: Despite potent inhibition of PKGI in vitro, (D)-DT-2 lost specificity for PKG in cell homogenates and particularly in living cells, as demonstrated by phosphorylation of different substrates. Instead, (D)-DT-2 modulated activity of other kinases including ERK, p38, PKB and PKC, thereby inducing unpredicted and often opposing functional effects. CONCLUSIONS AND IMPLICATIONS: We conclude that DT-oligopeptides, as other inhibitors, cannot be used to specifically inhibit PKG in intact cells. Therefore, no specific pharmacological PKG inhibitors are available, and reliable studies of PKG signalling can only be made by using RNA knockdown or genetic deletion methods.

The thrombin inhibitors hirudin and Refludan(®) activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets.

A number of direct thrombin inhibitors are successfully used clinically and experimentally as novel antithrombotics and specific anticoagulants. They are also used as anticoagulants in certain blood collection tubes for the analysis of platelet function. A series of platelet function tests have emerged to measure adequate responses to antiplatelet therapy. For comparative and practical reasons, it would be of advantage to use the same anticoagulant in blood collection tubes for different methods, e.g. thrombin inhibitors. However, there are little data on the effects of thrombin inhibitors on platelet signalling pathways that could influence results. We examined the applicability of thrombin inhibitor containing blood for platelet reactivity index (PRI) measurements of the VASP assay and investigated the effects of two thrombin inhibitors (hirudin and lepirudin) on cAMP- and cGMP-mediated signalling pathways in washed human platelets. We show that induction of VASP phosphorylation by PGE1 is markedly reduced in lepirudin containing blood samples. In consequence, PRI levels were highly variable compared to routinely used citrated blood. Surprisingly, in vitro incubation of platelets with thrombin inhibitors increases platelet cGMP levels and induces NOS independent sGC/PKG-mediated VASP phosphorylation. We conclude that thrombin inhibitors activate sGC/PKG-dependent pathways resulting in an increase of VASP phosphorylation which contributes to deviations in PRI measurements. These effects of thrombin inhibitors on sGC- and cGMP-mediated pathways including increased VASP phosphorylation may indicate the presence of an important additional platelet-based mechanism for the reduction of thrombus formation and thromboembolism by thrombin inhibitors.