Platelet Hemostatic Function Research Articles

The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.

Targeted Phosphotyrosine Profiling of Glycoprotein VI Signaling Implicates Oligophrenin-1 in Platelet Filopodia Formation

Platelet adhesion to subendothelial collagen is dependent on the integrin α2β1 and glycoprotein VI (GPVI) receptors. The major signaling routes in collagen-dependent platelet activation are outlined; however, crucial detailed knowledge of the actual phosphorylation events mediating them is still limited. Here, we explore phosphotyrosine signaling events downstream of GPVI with site-specific detail. Immunoprecipitations of phosphotyrosine-modified peptides from protein digests of GPVI-activated and resting human platelets were compared by stable isotope-based quantitative mass spectrometry. We surveyed 214 unique phosphotyrosine sites over 2 time points, of which 28 showed a significant increase in phosphorylation on GPVI activation. Among these was Tyr370 of oligophrenin-1 (OPHN1), a Rho GTPase-activating protein. To elucidate the function of OPHN1 in platelets, we performed an array of functional platelet analyses within a small cohort of patients with rare oligophrenia. Because of germline mutations in the OPHN1 gene locus, these patients lack OPHN1 expression entirely and are in essence a human knockout model. Our studies revealed that among other unaltered properties, patients with oligophrenia show normal P-selectin exposure and αIIbβ3 activation in response to GPVI, as well as normal aggregate formation on collagen under shear conditions. Finally, the major difference in OPHN1-deficient platelets turned out to be a significantly reduced collagen-induced filopodia formation. In-depth phosphotyrosine screening revealed many novel signaling recipients downstream of GPVI activation uncovering a new level of detail within this important pathway. To illustrate the strength of such data, functional follow-up of OPHN1 in human platelets deficient in this protein showed reduced filopodia formation on collagen, an important parameter of platelet hemostatic function.

The Small GTPase Rif Is Dispensable for Platelet Filopodia Generation in Mice

BackgroundFormation of filopodia and other shape change events are vital for platelet hemostatic function. The mechanisms regulating filopodia formation by platelets are incompletely understood however. In particular the small GTPase responsible for initiating filopodia formation by platelets remains elusive. The canonical pathway involving Cdc42 is not essential for filopodia formation in mouse platelets. The small GTPase Rif (RhoF) provides an alternative route to filopodia generation in other cell types and is expressed in both human and mouse platelets.Hypothesis/ObjectiveWe hypothesized that Rif might be responsible for generating filopodia by platelets and generated a novel knockout mouse model to investigate the functional role of Rif in platelets.Methodology/Principal FindingsConstitutive RhoF−/− mice are viable and have normal platelet, leukocyte and erythrocyte counts and indices. RhoF−/− platelets form filopodia and spread normally on various agonist surfaces in static conditions and under arterial shear. In addition, RhoF−/− platelets have normal actin dynamics, are able to activate and aggregate normally and secrete from alpha and dense granules in response to collagen related peptide and thrombin stimulation.ConclusionsThe small GTPase Rif does not appear to be critical for platelet function in mice. Functional overlap between Rif and other small GTPases may be responsible for the non-essential role of Rif in platelets.

Bleeding Risk after Invasive Procedures in Aspirin/NSAID Users: Polypectomy Study in Veterans

BackgroundAspirin, by virtue of inhibition of platelet hemostatic function, is withheld before many invasive procedures because of the bleeding risk. American Society of Gastrointestinal Endoscopy guidelines acknowledge the paucity of “high quality data” to make recommendations regarding the use of aspirin (ASA) and nonsteroidal anti-inflammatory drugs (NSAIDs) before endoscopic procedures. Yet the majority of endoscopists hold ASA/NSAIDs before polypectomy. MethodsThis single-center, retrospective, cohort study was conducted at Veterans Affairs Medical Center, Syracuse, NY. The objectives were to assess the postpolypectomy bleeding risk in ASA/NSAID users in a large cohort of veterans undergoing colonoscopic polypectomy and to identify risk factors associated with postpolypectomy bleeding. All patients undergoing polypectomy between January 2002 and October 2007 were eligible. Patients on anticoagulants/other antiplatelet agents were excluded. Patients were selected randomly by cluster sampling techniques. Electronic medical and pharmacy records were reviewed for patient demographics, polypectomy techniques, and postpolypectomy bleeding rates. Univariate analysis was performed between patients on ASA and NSAIDs (group A) versus those not on ASA or NSAIDs (group B). Multiple regression analysis was performed to identify independent risk factors associated with postpolypectomy bleeding. ResultsFive hundred two (43%) of 1174 patients (mean age 66 years, 92% white) were on ASA or NSAIDs, or both. There was no significant difference between postpolypectomy bleeding rates among the 2 groups (3.2% vs 3.0%). Age, sex, polyp characteristics, and polypectomy techniques were comparable between groups A and B. In multiple logistic regression analysis, ASA or NSAID use was not a significant risk factor for postpolypectomy bleeding. Number of polyps removed per patient was the only risk factor significantly associated with postpolypectomy bleeding (P <.01, odds ratio 1.3, 95% confidence interval, 1.18-1.43). ConclusionsIn this large study, use of ASA or NSAIDs did not increase the risk of postpolypectomy bleeding. Cessation of ASA/NSAIDs before colonoscopy/polypectomy is therefore unnecessary.

Bcl-xL–inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets

AbstractBH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2–related proteins. Agents that inhibit Bcl-xL induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin αIIbβ3. Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-xL–inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha‐granule proteins

Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures. To explore the effect of higher temperatures on platelet physiology. Platelet responses including adhesion, spreading (fluorescence microscopy), α(IIb)β(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied. Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion. Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.

Effects of multiwall carbon nano-onions on platelet aggregation and hemostatic function

To observe the effects of multiwall carbon nano-onions (MWCNOs) on platelet aggregation and hemostatic function. The platelet aggregation was determined with Born's method at different concentration of MWCNOs (0, 0.2, 2.0, 20.0 microg/ml) in vitro. Twenty male SD rats were randomly divided into 4 groups which were exposed to 0, 2, 4 and 8 mg/kg MWCNOs, respectively. Then platelet count, platelet aggregation, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), bleeding time (BT) and platelet count (PC) were measured at 12 h after receiving tail intravenous injection of MWCNOs. The effects of MWCNOs (4 mg/kg) on platelet aggregation and platelet count at different time points were observed. In vitro, MWCNOs exhibited the potent inhibitory effects on rat platelet aggregation caused by ADP in a concentration-dependent manner. The platelet aggregation in the highest dosage of 20.0 microg/ml group was 50.0% +/- 6.9% which was significantly lower than that (73.2% +/- 4.3%) in control group (P<0.01). In vivo, the highest inhibitory was up to 20.4%, but there was no significant difference, as compared with control group. MWCNOs did not affect the APTT, PT, TT, BT and PC. Under this experimental condition, MWCNOs might inhibit platelet aggregation but not affect hemostatic function.

P38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

AbstractPlatelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the alpha(2)beta(1) integrin (alpha(2)beta(1)) and the glycoprotein VI (GPVI)/FcRgamma chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (alpha2-CRP) containing the alpha(2)beta(1)-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to alpha2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was alpha(2)beta(1)-dependent and GPVI/FcRgamma-independent as revealed in experiments with alpha(2)beta(1)- or FcRgamma-deficient mouse platelets. We further show that suboptimal activation of other platelet G(q)-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to alpha2-CRP. The enhanced alpha(2)beta(1)-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of alpha(IIb)beta(3) integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G(q)-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced alpha(2)beta(1) binding to collagens containing GFOGER sites.

New insights into the haemostatic function of platelets

Considerable progress has been made over the last two decades in delineating the key molecular events regulating the haemostatic function of platelets. Much of this new insight has been derived from the study of mouse models, in which the expression or structure of one or more platelet proteins has been genetically altered. Despite these advances on the research front, clinical progress in diagnosing patients with unexplained surgical bleeding or recurrent haemorrhage from mucocutaneous sites has been comparatively limited. There is a dearth of literature available to help physicians integrate and apply the burgeoning knowledge on platelet biology to diagnosing patients with atypical or unexplained platelet dysfunction. The purpose of this review is to summarise the major primary platelet disorders relevant to pathological bleeding in humans (excluding those primarily due to thrombocytopenia or acquired functional disorders), with a focus on lesions identified in mouse models that could represent candidate molecules for study in patients with impaired platelet function.

A025 P38 MAPK inhibition prevents TACE-mediated receptor shedding and improves the hemostatic function of stored platelets

Platelet transfusion is a lifesaving procedure widely used during surgery and chemotherapy. During storage platelets undergo several modifications that reduce their postransfusion survival and functionality. One important feature of this platelet storage lesion (PSL) is the shedding of surface glycoproteins such as GPIbα and GPV. We have recently demonstaretd that metalloproteinase inhibitors prevent storage-induced shedding of adhesion receptors, resulting in markedly improved postransfusion recovery and hemostatic function of platelets in mice. We now demonstrate that TNF-alpha converting enzyme (TACE/ADAM17) mediates receptor shedding during platelet storage for 16 to 18 hours at 37oC or 22oC. Using pharmacological inhibitors, we demonstrate that TACE/ADAM17-dependent shedding of GPIbα and GPV from both mouse and human platelets during storage required p38 MAP kinase signaling. In contrast, protein kinase C, Erk MAPK, and caspases were not involved. Inhibition of p38 MAPK during platelets storage also markedly improved the posttransfusion recovery and hemostatic function of stored platelets in vivo. Moreover, p38 MAPK inhibition during storage of human platelets improved the adhesion of these cells to collagen under flow. Inhibition of p38 MAPK also prevented TACE-dependent shedding of GPIbα from platelets undergoing mitochondrial injury, a model for PSL. Phosphorylation of p38 MAPK was observed upon platelet storage and mitochondrial injury. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

P38 MAPK Inhibition Prevents TACE-Mediated Receptor Shedding and Improves the Hemostatic Function of Stored Platelets.

Platelet transfusion is a lifesaving procedure widely used during surgery and chemotherapy. During storage platelets undergo several modifications that reduce their postransfusion survival and functionality. One important feature of this platelet storage lesion (PSL) is the shedding of surface glycoproteins such as GPIbα and GPV. We have recently demonstaretd that metalloproteinase inhibitors prevent storage-induced shedding of adhesion receptors, resulting in markedly improved postransfusion recovery and hemostatic function of platelets in mice. We now demonstrate that TNF-alpha converting enzyme (TACE/ADAM17) mediates receptor shedding from platelet stored for 18 hours at 37ºC or 22ºC. Using pharmacological inhibitors, we show that TACE -dependent shedding of GPIbα and GPV from both mouse and human platelets during storage required p38 MAP kinase signaling. In contrast, protein kinase C, Erk MAPK, and caspases were not involved. Inhibition of p38 MAPK during the storage of mouse platelets also markedly improved their posttransfusion recovery and hemostatic function in vivo. Moreover, p38 MAPK inhibition during storage of human platelets improved their adhesion to collagen under flow. Inhibition of p38 MAPK also prevented TACE-dependent shedding of GPIbα from platelets undergoing mitochondrial injury, a model for PSL. Phosphorylation of p38 MAPK was observed after platelet storage and mitochondrial injury. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Von Willebrand disease (VWD): evidence‐based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA)1

von Willebrand disease (VWD) is a commonly encountered inherited bleeding disorder affecting both males and females, causing mucous membrane and skin bleeding symptoms, and bleeding with surgical or other haemostatic challenges. VWD may be disproportionately symptomatic in women of child-bearing age. It may also occur less frequently as an acquired disorder (acquired von Willebrand syndrome). VWD is caused by deficiency or dysfunction of von Willebrand factor (VWF), a plasma protein that mediates platelet haemostatic function and stabilizes blood coagulation factor VIII. The pathophysiology, classification, diagnosis and management of VWD are relatively complex, but understanding them is important for proper diagnosis and management of patients with VWD. These evidence-based guidelines for diagnosis and management of VWD from the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel (USA) review relevant publications, summarize current understanding of VWD pathophysiology and classification, and present consensus diagnostic and management recommendations based on analysis of the literature and expert opinion. They also suggest an approach for clinical and laboratory evaluation of individuals with bleeding symptoms, history of bleeding or conditions associated with increased bleeding risk. This document summarizes needs for further research in VWF, VWD and bleeding disorders, including clinical research to obtain more objective information about bleeding symptoms, advancements in diagnostic and therapeutic tools, and enhancement in the education and training of clinicians and scientists in bleeding and thrombotic disorders. The NHLBI Web site (http://www.nhlbi.nih.gov/guidelines/vwd) has a more detailed document, a synopsis of these recommendations, and patient education information.

Markers of platelet activation and apoptosis in platelet concentrates collected by apheresis

Background A product with well-preserved haemostatic function of platelets is the ultimate goal of platelet concentrate production. However, platelet activation and apoptosis are induced by both collection and storage of platelet concentrates. Aim of study Platelet concentrates obtained either by two blood separators with different technology of apheresis (Haemonetics MCS+, Haemonetics Corp. Braintree, USA and Trima Accel, Gambro BCT Inc., Lakewood, USA, respectively) or derived from buffy-coat were compared using evaluation of pH, LDH, lactate, glucose, annexin V, and sP-selectin levels immediately after collecting and at the end of expiration to estimate the differences in the activation and apoptosis of platelets in these products. Results The lowest degree of platelet activation was found in products obtained by Haemonetics MCS+ apparatus at the time of collection. Platelet concentrates obtained by apheresis revealed higher rise of LDH, annexin V and sP-selectin compared to buffy-coat derived platelets. Products from Haemonetics MCS+ showed higher rise of annexin V in comparison with products from Trima separator. Increase of LDH and sP-selectin in both apheresis products was comparable. Conclusions On the basis of changes of sP-selectin and annexin V levels it could be concluded that initial platelet activation, which is induced by apheresis, is very likely without any further impact on quality of platelets during storage. Development of platelet storage lesions is influenced especially by storage conditions and platelet concentration in products.

Rapid and reversible modulation of platelet function in man by a novel P2Y12 ADP-receptor antagonist, INS50589

P2Y12 receptors participate in ADP-induced activation and aggregation of human platelets. INS50589, a selective P2Y12 receptor antagonist, is being developed for use where controlled, reversible modulation of the platelet hemostatic function is needed. The tolerability, pharmacokinetics, and pharmacodynamics of INS50589 were tested in healthy human volunteers. Thirty-six subjects received intravenous infusions of placebo or INS50589 at 0.1–3 mg/kg/h for four hours. Platelet function, clotting parameters, bleeding time, safety assessments, and plasma concentrations of INS50589 and its major metabolite were monitored for 24 hours. Near-steady state plasma concentrations of INS50589 and effects on platelet function were achieved rapidly. The average maximal plasma concentration of INS50589 was linearly related to the dose administered. Intravenous INS50589 produced dose-dependent inhibition of platelet activation and aggregation in response to ADP in vitro until nearly full inhibition was achieved at the higher doses. Bleeding time was correspondingly increased, without any effect on activated clotting time, prothrombin time, or activated partial thromboplastin time. Platelet response to ADP had returned to at least 75% of the baseline value within 0.25–4 h after cessation of the intravenous infusion of INS50589, depending upon the dose and ADP challenge concentration. Infusions were well tolerated up to the highest dose tested. There was no evidence that the principal metabolite (INS51088) contributed to these effects. INS50589 is a well-tolerated, reversible, competitive antagonist of ADP at the P2Y12 human platelet receptor, and its potential therapeutic utility in various cardiovascular settings is discussed.

Platelet Proinflammatory and Hemostatic Function Is Differentially Regulated in Cystic Fibrosis.

Introduction: Platelet function and mechanisms of platelet-leukocyte interactions have been investigated in several vascular und inflammatory disorders. In most studies, platelet activation and an increase of platelet-leukocyte-aggregates (PLA) could be observed. We investigated platelet function in clinically stable patients with cystic fibrosis (CF).Methods: In addition to routine markers of inflammation (e. g. CRP, IgG, ESR) parameters of platelet function were measured in 54 clinically stable CF patients and 55 healthy controls (age range 3 to 41 years): The percentage of P-selectin (CD62P) and PAC-1 (activated integrin αIIbβ3) positive resting and activated platelets (in-vitro activation with the thrombin receptor activating peptide 6) and the number of PLA were determined by flow cytometry. The plasma markers of platelet activation soluble P-selectin (sCD62P) and soluble CD40 ligand (sCD40L) were measured by ELISA. Furthermore, 15 CF patients and 14 healthy controls were investigated to determine CD41a-expression (integrin αIIbβ3) on resting and activated platelets as well as leukocyte expression of P-selectin-glycoprotein ligand 1 (PSGL-1, receptor of CD62P) and integrin αMβ2.Results: Chronic inflammation leads to a decrease of PAC-1-binding to resting and activated platelets. The effects were stronger in patients with higher markers of inflammation. CD41a expression was reduced on in-vitro-activated CF platelets. In contrast, proinflammatory platelet functions remained unchanged (CD62P-expression on resting and activated platelets) or increased (sCD62P, sCD40L, PLA). Leukocyte integrin αMβ2 expression was increased and PSGL-1 expression remained unchanged in CF.Discussion: Platelet function in clinically stable patients with CF is differentially regulated: Chronic inflammation leads to an upregulation of platelet proinflammatory function. In the presence of several procoagulatory mechanisms in inflammation there is a compensatory loss of platelet hemostatic function, shown by the decreased activation and exocytosis of the platelet major integrin αIIbβ3.

The Mechanism of Cold-Induced Platelet Hyperfunction.

Refrigerated platelets function better when transfused than do platelets stored at room temperature, although chilled and rewarmed platelets do not survive long in the circulation. Here, we describe the one possible mechanism of this increase in the hemostatic function of platelets upon cold treatment. Cold exposure has been demonstrated to cluster GP Ib-IX complexes on platelets, enabling platelet recognition and phagocytosis by macrophages. We find that, in addition to increasing the binding to the leukocyte integrin Mac-1 (aMb2), clustering of GP Ib-IX complexes also augments von Willebrand factor (VWF)-related functions. Before the functional studies, platelets were chilled to 4°C for 2 hr and rewarmed to 37°C for 30 min. Chilled, rewarmed platelets, whether in platelet-rich plasma (PRP), whole blood, or reconstituted whole blood (all anticoagulated with Cirtate Sodium) perfused through a parallel-plate flow chamber (36 dynes/cm2) attached in greater numbers to VWF-coated surfaces than did platelets from the same donor kept at 37°C or 22°C. The increased function was also reflected in increased thrombus number and size on a surface of type III collagen. The surface coverage, an indicator of thrombus formation, increased from 10% to 25% upon cold. Chilled, rewarmed platelets also responded to lower doses of a VWF-specific agonist, ristocetin Chilled, rewarmed platelets showed 80% aggregation while control platelet only had 10% aggregation at low concentration of ristocetin (0.5mg/ml) treatment, and displayed a decreased threshold for shear-induced aggregation, aggregating at lower shear rates than control platelets at both 22°C and 37°C. Finally, when tested in a platelet function analyzer (PFA-100), chilled, rewarmed platelets demonstrated significantly shorter closure times than control platelets using both epinephrine/collagen and ADP/collagen cartridges. The epinephrine/collagen cartridges clotting time of chilled platelets decreased from normal 180 s to 162 s and ADP/collagen cartridges clotting time decreased from 118s to 99s. To exclude that the observed functional increases were not due to changes in GP Iba expression or prior platelet activation, we evaluated by flow cytometry the surface levels of GP Iba, P-selectin, and activated αIIbβ3.(antibody PAC-1 binding). We found no differences in any of these parameters between chilled, rewarmed platelets and control platelets. These results suggest that, if their rapid clearance can be prevented, cold-stored platelets may have enhanced hemostatic function of potential benefit in the bleeding patient. The results also caution, however, that trials will be necessary to exclude potential thrombotic consequences of infusing hyperfunctional, refrigerated platelets.

Inhibition of the Binding of von Willebrand Factor to the Platelet Glycoprotein Ib-IX with an Inhibitor of 14-3-3 and Glycoprotein Ib-IX Interaction.

Platelet adhesion and activation are essential for thrombosis and hemostasis. In arteries and capillaries where blood flow shear rate is high, initial platelet adhesion is dependent on the binding of von Willebrand factor (VWF) to its platelet receptor, the glycoprotein (GP) Ib-IX-V complex (GPIb-IX), which consists of four subunits, GPIbα, GPIbβ, GPIX and GPV. We have shown previously that a phosphoserine-dependent intracellular signaling molecule, 14-3-3, interacts with the C-terminal SIRYSGHSL610 sequence in the cytoplasmic domain of GPIbα, and this interaction is dependent upon phosphorylation at Serine 609 of GPIbα. Here we show that a short cell-permeable myristoylated phospho-peptide corresponding to the 14-3-3 binding sequence of GPIbα inhibits VWF binding to platelets and VWF-mediated platelet adhesion. This peptide also specifically inhibits VWF-dependent platelet agglutination induced by ristocetin but has no significant effect on platelet aggregation induced by platelet agonists such as ADP and collagen. Furthermore, intravenous injection of this phospho-peptide caused significantly prolonged bleeding time in mice. Thus, 14-3-3 interaction with GPIbα plays critical roles in VWF binding function of GPIb-IX and hemostatic function of platelets. These results also suggest a new type of anti-platelet drugs that may potentially be useful in treating thrombosis.

Profiles of Antibodies Specific to Platelet Glycoproteins in ITP Differ in Remission, Relapse and Stable Phases; and Anti-GpIIb/IIIa, -GpIb/IX Were Associated with Bleeding Manifestations.

BACKGROUND: In immune thrombocytopenic purpura (ITP), antiplatelet antibodies (APtAb) bind to antigens exposed on the platelet surface to mediate platelet clearance from circulation. GpIIb/IIIa, GpIb/IX and GpIV, common target antigens in ITP, are receptors for fibrinogen, von Willebrand factor and collagen, respectively, and are involved in platelet aggregation and adhesion, playing critical roles in hemostasis and thrombosis. It has been suggested that APtAb binding to these molecules interfere with their function, inducing platelet dysfunction and enhancing bleeding in ITP. In the present study, we investigated profiles of antibodies against these antigens in different stages of ITP, and in relation to bleeding manifestations.METHODS: We measured IgG and IgM APtAb against the three most prevalent glycoprotein target antigens, GpIIb/IIIa, GpIV and GpIb/IX, by PAICA [Macchi, et al; 1996, Thromb Haemost 76:1820]. We studied 130 patients with ITP. There were 87 women (66.9%) and 43 men (33.1%). ITP was classified into three stages, (i) acute/relapse, 28 pts (21.5%); (ii) chronic/stable (platelet counts <150,000 for at least 6 mo's), 69 pts (53.0%); (iii) remission, 33 pts (25.3%). The chronic/stable patients were further divided into bleeders (n=27) and non-bleeders (n=19). Those patients with visible multiple petechias or echymosis at more than three sites, or mucosal bleeding were defined as bleeders; non-bleeders had none of these signs.RESULTS: (1) IgG APtAb against GpIIb/IIIa, GpIV and GpIb/IX were all significantly more frequent in acute/relapse (77%, 70%, and 67%, respectively) than in chronic/stable ITP (52%,55%, and 44%), at p=0.005, p=0.003, p=0.04, respectively. (2) All IgG APtAb were significantly more frequent in acute/relapse and chronic/stable ITP compared to remission, p<0.0001. (3) IgM APtAb were detected in approximately 50% of patients in relapse and stable stages for all 3 antigens; there was no significant difference in frequency between acute/relapse and chronic/stable, but both were significantly higher than in remission. (4) Multiple antibodies (≥ 2 antigens positive) were observed in 50–70% of patients in both relapse and stable stages. (5) When we compared frequencies of APtAb between bleeders and non-bleeders, both IgG and IgM APtAb were significantly more frequent in bleeders, for all antigens except IgM GpIb/IX. Interestingly, 96.7% of bleeders had at least one APtAb of IgG or IgM class, while only 58% were positive in non-bleeders (p<0.0002). This difference persisted when reanalyzed to ensure equal mean platelet counts between bleeders and non-bleeders. It was also found that positivity for APtAb of IgG (but not IgM) class against the pair, GpIIb/IIIa and GpIb/IX (but not GpIV) was predictive of bleeding.CONCLUSIONS. Distinctive profiles of APtAb to GpIIb/IIIa, GpIb/IX, and GpIV were observed in different stages of ITP. IgG APtAb were frequently seen in acute/relapse and chronic/stable, both clases (IgG-IgM) were rarely seen in remission. Patients with signs of bleeding more frequently had IgG APtAb to the pair, GpIIb/IIIa and GpIb/IX, suggesting this pair of APtAb interferes with platelet hemostatic functions, enhancing the bleeding manifestations of ITP.

Leucocyte-Reduced Buffy Coat Platelet Concentrates (BCPC) Treated with INTERCEPT™ Stored up to 7 Days: Hemostatic Function in a Whole Blood Flow System.

Background: The risk of bacterial growth has limited the shelf life of platelet concentrates (PC) to 5 days. Modern platelet storage containers facilitate storage for up to 7 days, if bacterial contamination is prevented. INTERCEPT (Baxter, La Chatre, France; Cerus, Concord, CA) photochemical treatment (PCT) for pathogen reduction based on amotosalen (150μM) and UVA illumination (3 J/cm2) inactivates high titers of bacteria in PC (Transfusion 2004; 44: 1496–1504). Adhesion and aggregation of platelets to injured vascular surfaces are critical aspects of platelet hemostatic function. In this study, the adhesion and aggregation of leucocyte-reduced buffy coat derived PC (BCPC), treated with INTERCEPT and stored up to 7 days, were measured on injured vascular surfaces using an ex-vivo blood flow system.Methods: BCPC were prepared from 450 mL-whole blood donations with the top and bottom method (Optipress II, Baxter). Five BCPC, of the same ABO group, were pooled with additive solution (Intersol™) the day following collection, after viral screening testing was completed. Following centrifugation and leukocyte depletion, two BCPC pools of the same ABO group were mixed and divided. One pooled BCPC was treated with INTERCEPT (I-BCPC) and the other was prepared by conventional methods (C-BCPC); and both were stored in 1.3 liter PL2410 plastic containers (Baxter R4R7012) at 22 ± 2°C with continuous agitation for 7 days. Samples for hemostatic function testing were taken immediately after preparation before splitting for treatment and after 5 and 7 days of storage. Platelet counts were performed in K3EDTA in a Coulter MD II counter (Coulter, Miami, FL). Samples of I-BCPC and C-BCPC were added to citrate anticoagulated blood, previously depleted of platelets and leukocytes by filtration, and adjusted to a final platelet count of 150x109/L. Enzymatically denuded vascular segments were exposed to circulating whole blood, reconstituted with I-BCPC and C-BCPC, in Baumgartner chambers at a shear rate of 800 s−1 for 10 minutes. The proportion (%) of the vascular surface area covered by platelets after perfusion was measured for each type of BCPC (N = 9) at each storage time point. Platelets and groups of platelets were classified as adhesive when platelet masses were less than 5 μm in height and as thrombi when height exceeded 5 μm. Data were analyzed with the SPSS 12.0.1 statistical package with significance at p < 0.05, and expressed at the mean ± SEM Results(Table).Conclusion: The platelet count of I- BCPC decreased by 12.3% including PCT processing losses and 7 days of storage. However, I- BCPC platelet adhesive and aggregatory capacities under flow conditions were similar to C- BCPC, and were well conserved for up to 7 days of storage.Hemostatic Function of Stored I-BCPC and C-BCPCParameterI-BCPCC-BCPCpDay 1(Pre Treatment)Platelet Count (109/L)945±40945±40Platelet Coverage (%)26.0±3.726.0±4.2Adhesion(%)24.0±3.724.0±3.7Thrombus(%)1.9±0.61.9±0.6Day 5 StoragePlatelet Count (109/L844±41902±440.004Platelet Coverage (%)20.9±2.220.6±1.60.9Adhesion(%)19.9±2.119.3±1.40.8Thrombus(%)0.9±0.31.2±0.40.5Day 7 StoragePlatelet Count (109/L)829±32923±480.008Platelet Coverage (%)27.1±2.921.2±2.80.06Adhesion(%)26.0±2.720.4±2.70.06Thrombus(%)1.2±0.30.7±0.20.16