Platelet surface receptor glycoprotein VI-dimer is overexpressed in stroke: The Glycoprotein VI in Stroke (GYPSIE) study results.

OBJECTIVES: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. METHODS: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). RESULTS: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). CONCLUSIONS: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood.

Objectives Analysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy. Methods In addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel. Results Established reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard-Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts. Conclusions This study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

Atherosclerosis, myocardial infarction and primary hemostasis: Impact of platelets, von Willebrand factor and soluble glycoprotein VI.

INTRODUCTION: Little is known about peril constellations in primary hemostasis contributing to an acute myocardial infarction (MI) in patients with already manifest atherosclerosis. The study aimed to establish a predicting model based on six biomarkers of primary hemostasis: platelet count, mean platelet volume, hematocrit, soluble glycoprotein VI, fibrinogen and von Willebrand factor ratio. MATERIALS AND METHODS: The biomarkers were measured in 1.491 patients with manifest atherosclerosis of the Leipzig (LIFE) heart study. Three groups were divided: patients with coronary artery disease (900 patients) and patients with atherosclerosis and either ST-elevated MI (404 patients) or Non-ST-elevated MI (187 patients). Correlations were analyzed by non-linear analysis with Self Organizing Maps. Classification and discriminant analysis was performed using Learning Vector Quantization. RESULTS AND CONCLUSIONS: The combination of hemostatic biomarkers is regarded as valuable tool for identifying patients with atherosclerosis at risk for MI. Nevertheless, our study contradicts this belief. The biomarkers did not allow to establish a predicting model usable in daily patient care. Good specificity and sensitivity for the detection of MI was only reached in models including acute phase parameters (specificity 0,9036, sensitivity 0,7937 in men; 0,8977 and 0,8133 in women). In detail, hematocrit and soluble glycoprotein VI were significantly different between the groups. Significant dissimilarities were also found for fibrinogen (in men) and von Willebrand factor ratio. In contrast, the most promising parameters mean platelet volume and platelet count showed no difference, which is an important contribution to the controversy concerning them as new risk and therapy targets for MI.

Device-induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding.

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device-induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4 hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P-selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device-injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device-induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4 hours) and soluble P-selectin concentration increased from 14.1 ng/mL (Base) to 26.5 ng/mL (4 hours). Those device-activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4-hours sample compared to the baseline samples, respectively. The device-injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device-altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device-induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device-induced loss of HMWM-VWF.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.

Switching from abacavir to tenofovir disoproxil fumarate is associated with rises in soluble glycoprotein VI, suggesting changes in platelet-collagen interactions.

OBJECTIVES: Altered platelet function has been proposed as an underlying mechanism to explain increased risk of myocardial infarction in people living with HIV associated with use of the nucleoside reverse transcriptase inhibitor abacavir (ABC). We aimed to examine changes in platelet biomarkers in people living with HIV switching from ABC. METHODS: In a prospective, 48-week substudy of virally suppressed HIV-1-positive subjects randomized to remain on ABC/lamivudine (ABC/3TC) or switch to tenofovir disoproxil fumarate/emtricitabine, we measured soluble glycoprotein VI (sGPVI), soluble P-selectin, soluble CD40 ligand and von Willebrand factor in plasma collected over time and assessed differences using mixed effect models. RESULTS: Of 312 randomized participants, 310 were included in the analysis. Mean (SD) age 46.4 (9.3) years, 262 (85%) men and 201 (65%) white. At baseline, there was no significant between-group difference in sGPVI [tenofovir disoproxil fumarate/emtricitabine 3.75 (0.25) versus ABC/3TC 3.61 (0.22) ng/ml, P = 0.69]. Greater increases in sGPVI from baseline to week 48 occurred in those switched from ABC/3TC (effect size +0.57 ng/ml; 95% confidence interval, 0.2-0.94; P = 0.003). There was no significant baseline difference or change overtime in soluble P-selectin, soluble CD40 ligand or von Willebrand factor between groups. CONCLUSION: The significant increases in sGPVI that occur with a switch from ABC/3TC are suggestive of changes in platelet function centred on platelet/collagen interactions and potentially represent an underlying mechanism to explain increased risk of myocardial infarction with ABC.

Association of Oxidative Stress and Platelet Receptor Glycoprotein GPIbα and GPVI Shedding During Nonsurgical Bleeding in Heart Failure Patients With Continuous-Flow Left Ventricular Assist Device Support.

Nonsurgical bleeding (NSB) in heart failure (HF) patients with continuous-flow left ventricular assist device (CF-LVAD) support is the most common clinical complication. The aim of this study was to investigate the association between oxidative stress and platelet glycoproteins GPIbα and GPVI shedding on the incidence of NSB in CF-LVAD patients. Fifty-one HF patients undergoing CF-LVAD implantation and 11 healthy volunteers were recruited. Fourteen patients developed NSB (bleeder group) during 1 month follow-up duration, while others were considered nonbleeder group (n = 37). Several biomarkers of oxidative stress were quantified at baseline and weekly intervals in all patients. Surface expression and plasma elements of platelet receptor glycoproteins GPIbα and GPVI were measured. Oxidative stress biomarkers and platelet GPIbα and GPVI receptor-shedding (decreased surface expression and higher plasma levels) were found to be preexisting conditions in baseline samples of both groups of HF patients when compared with healthy volunteers. Significantly elevated oxidative stress biomarkers and platelet glycoprotein receptor shedding were observed in postimplant bleeder group temporarily when compared with nonbleeder group. Strong significant associations between biomarkers of oxidative stress and platelet glycoprotein receptor shedding were observed, suggesting a possible role of oxidative stress in platelet integrin shedding leading to NSB in CF-LVAD patients. Receiver operating characteristic analyses of GPIbα and GPVI indicated that the likelihood of NSB had a predictive power of bleeding complication in CF-LVAD patients. In conclusion, elevated oxidative stress may play a role in GPIbα and GPVI shedding in the event of NSB. Thus, oxidative stress and GPIbα and GPVI shedding may be used as potential biomarkers for bleeding risk stratification in those patients.

Platelet Activation Assessed by Glycoprotein VI/Platelet Ratio Is Associated With Portal Vein Thrombosis After Hepatectomy and Splenectomy in Patients With Liver Cirrhosis.

Portal vein thrombosis (PVT) is a serious complication after hepatobiliary-pancreatic surgery. Portal vein thrombosis often develops in patients with liver cirrhosis (LC) postoperatively, although they have low platelet counts. Platelet activation is one of the causes of thrombosis formation, and soluble form of glycoprotein VI (sGPVI) has received attention as a platelet activation marker. We had prospectively enrolled the 81 consecutive patients who underwent splenectomy (Sx) and/or hepatectomy: these patients were divided as Sx (n = 38) and hepatectomy (Hx, n = 46) groups. The 3 patients who underwent both procedures were added to both groups. Each group was subdivided into patients with non-LC and LC: non-LC-Sx (n = 22) and LC-Sx (n = 16), non-LC-Hx (n = 40) and LC-Hx (n = 6). The presence of PVT was diagnosed by using enhanced computed tomography (CT) scan. Platelet counts were significantly lower in LC-Sx than in non-LC-Sx, and incidence of PVT was significantly higher in LC-Sx than in non-LC-Sx (68.8% vs 31.8%, P = .024). Soluble form of glycoprotein VI /platelet ratios on preoperative day and postoperative day 1 were significantly higher in LC-Sx than in non-LC-Sx. Incidence of PVT was significantly higher in LC-Hx than in non-LC-Hx (50.0% vs 7.5%, P < .01). Soluble form of glycoprotein VI /platelet ratios were significantly higher in LC-Hx before and after Hx, compared to non-LC-Hx. Patients with LC stay in hypercoagulable state together with platelet activation before and after surgery. Under this circumstance, alteration of portal venous blood flow after Sx or Hx is likely to cause PVT in patients with LC.

Flow Cytometry Protocols for Assessment of Platelet Function in Whole Blood.

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.

[The preparation and evaluation of the quality control materials for detection of platelet membrane glycoproteins by flow cytometry].

Objective: To prepare the quality control material for detection of platelet membrane glycoproteins by flowcytometry and evaluate the appearance traits, homogeneity and stability of it. Methods: Fresh platelets from the blood group O donors were fixed by the certain concentration of aldehyde solution and then washed by the imidazole buffer. After that, adding certain concentration of lyophilized protection solution into the preparations. The preparations were dispensed to be lyophilized and then were kept refrigerated in 2-8 ℃.According to the protocol of control of lyophilized biological products, the quality indicator for monitoring the prepared process, containing the appearance traits, the residual water, the platelet recovery and the rehydration quality were evaluated. The homogeneity and stability of these preparations were evaluated according to the CNAS-GL03 Guidance on evaluating the homogeneity and stability of samples used for proficiency testing and the ISO Guide 35 Reference material-general and statistical principles for certification. Results: The appearance traits and the rehydration quality of the quality control materials meeted the requirements, with the residual water distributed between 3.96% to 4.04% and the platelet recovery rate ranged from 68% to 72%.The homogeneity evaluation showed that there was no significant difference among the groups(P>0.05). The stability test indicated that the positive rate of platelet membrane glycoproteins CD42b, CD41 and CD62P of the quality control material was -0.14%, -0.14% and 0.74%, respectively, at 16 weeks after storage. There was no linear trend between the percentage of positive platelets with membrane glycoproteins and time(P>0.05). Conclusions: The quality control material for detection of platelet membrane glycoproteins by flow cytometry prepared by us meets the needs of the appearance traits, the residual water, the rehydration quality, the homogeneity and the longtime stability.It is hopeful to be used as internal quality control of the assay in clinic laboratory, the external quality assessment and proficiency evaluation.

Platelet reactivity and mean platelet volume as risk markers of thrombogenesis in atrial fibrillation.

Atrial fibrillation (AF) is associated with increased risk of thromboembolic complications. One of the markers of the increased risk of hypercoagulable state is platelet hyperreactivity. The aim of the study was to assess impact of arrhythmia on platelet reactivity. METHODS: The study included 36 (mean age 48,3; range 21-60) male patients with lone atrial fibrillation, with exclusion of concomitant diseases known to trigger hypercoagulable state. The AF patients underwent cardioversion to restore sinus rhythm and were subsequently under observation for 1month. Echocardiography, ECG and blood collection was performed before cardioversion (T0) and 4weeks after successful cardioversion (T1). During the study period patients have been contacted and examined every week and 24h ECG monitoring was performed. Platelet reactivity was assessed based on changes of CD62 and CD42b expression on platelet surface after stimulation with thrombin. Also changes in MPV were assessed. RESULTS: In all patients sinus rhythm was maintained at the end of the study period, however in 14 patients recurrences of AF were observed, confirmed by 24h ECG monitoring (atrial fibrillation recurrence group - AFR) and 22 patients maintained sinus rhythm throughout the whole study period (SR group). Mean fluorescence intensity (MFI) of CD62 on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (48.04±22.42 vs 41.47±16.03; p<0.01). Also MFI of CD42b on thrombin stimulated platelets decreased significantly 4weeks after electrical cardioversion as compared to T0 (22.16±10.82 vs 12.06±5.99; p<0.0001). Platelets reactivity estimated by CD 62 expression in SR group decreased significantly after 4weeks observation (58.01±15.26 vs 46.57±13.44; p<0.001) opposite to AFR group 35.66±21.87 vs 34.54±16.4; p-ns). Moreover there were significant differences between basal reactivity during AF between SR and AFR groups (58.01±15.26 vs 35.66±21.87; p-0.01). MFI of CD42b on thrombin stimulated platelets decreased significantly both in AFR and SR groups (22.05±11.36 vs 13.8±6.03; p<0.001 and 21.87±14.18 vs 10.04±5.09; p<0005). MPV decreased significantly 4weeks after electrical cardioversion as compared to T0 (8.81±0.19 vs 8.42±0.14; p<0.0001). CONCLUSION: The changes of platelet reactivity to thrombin observed after restoration of sinus rhythm in patients prove that arrhythmia intrinsically leads to increased reactivity of platelets.

Elevated Soluble Platelet Glycoprotein VI Levels in Patients After Living Donor Liver Transplantation.

Plasma-soluble platelet glycoprotein VI (sGPVI) levels were examined in patients undergoing living donor liver transplantation (LDLT), and the relationship between platelet activation and thrombocytopenia was evaluated to understand the mechanism of thrombocytopenia in LDLT. Platelet counts were significantly higher in the donors compared to the recipient, and the plasma sGPVI levels increased in both groups after the operation. Regarding the relationship between the platelet counts and the sGPVI levels, the slope varied on different days, and it became negative on day 3, suggesting that the plasma sGPVI levels are related to platelet activation in LDLT. The frequency of complications was high in the nonsurvivors. The platelet counts were higher in the survivors than in the nonsurvivors on days 14 and 28. Although the plasma levels of sGPVI in the survivors increased after the operation, those in the nonsurvivors were high only on day 3. Although the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 domain, member 13) levels were markedly reduced, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were markedly elevated during LDLT. The antithrombin activity was significantly lower (day 14) and VWFpp (day 28) was significantly higher in the nonsurvivors than in the survivors. These findings suggest that platelet activation first occurs after LDLT, and it is high in the nonsurvivors on day 3. Thereafter, the hemostatic abnormality and vascular endothelial cell injuries may appear on days 14 and 28.

Increased soluble GPVI levels in cirrhosis: evidence for early in vivo platelet activation.

Cirrhosis is a consequence of prolonged liver injury and is characterised by extensive tissue fibrosis: the deposition of collagen-rich extracellular matrix. The haemostatic balance is disordered in cirrhosis and coagulation activation appears to promote fibrosis. In spite of recent studies demonstrating a role for anticoagulant therapy in preventing cirrhosis progression, there has not been a change in clinical practice, suggesting that physicians are reluctant to anticoagulate patients with cirrhosis due to bleeding risks. Platelets play an important role in facilitating coagulation. Glycoprotein VI (GPVI) is a platelet-specific collagen receptor that is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligation and coagulation activation. Our aim was to use soluble GPVI levels to determine whether there was evidence for collagen and coagulation-induced platelet activation in early, well-compensated cirrhosis. Plasma soluble GPVI levels were quantified in 46 patients with mixed aetiology cirrhosis and 55 healthy controls using an immunoassay. In the cirrhosis group, soluble GPVI levels were significantly increased (5.8 ± 4.4 ng/ml, n = 46) compared to healthy controls (3.3 ± 3.4 ng/ml, n = 55, p < 0.05). This increase in soluble GPVI levels was still evident when levels were adjusted for platelet count (Healthy controls; 0.015 ± 0.018 ng/106 platelets/ml vs. cirrhosis; 0.048 ± 0.04 ng/106 platelets/ml, p < 0.0001). This study provides evidence for early platelet activation in patients with well-compensated cirrhosis. This may have translational implications for prognosis, treatment, and risk stratification.

Evaluation of soluble glycoprotein V as an in vitro quality marker for platelet concentrates: a correlation study between in vitro platelet quality markers and the effect of storage medium.

BACKGROUND AND OBJECTIVES: Extent of shape change (ESC), hypotonic shock response (HSR), the adhesive glycoprotein CD62P and lactate have been widely used as in vitro quality makers of platelet concentrates. Our aim was to evaluate soluble glycoprotein V (sGPV) as a platelet in vitro activation marker for platelet concentrates. Data were obtained from different validations during a twelve-year period. MATERIALS AND METHODS: Platelet concentrates stored in PAS B, PAS E with 20-35% plasma carry-over or in plasma were prepared either from buffy coats or collected by apheresis. The samples were analysed up to day seven using sGPV, ESC, HSR, CD62P and lactate in addition to normal quality control assays. RESULTS: sGPV correlated statistically significantly with ESC, HSR, CD62P and lactate in platelet concentrates stored in PASs. CONCLUSION: We propose that quantitative sGPV which is calculated per platelet number could be used as the first choice in vitro platelet activation marker in validations and monitoring platelet production processes. sGPV could also be a promising alternative to platelet activation assays used in interlaboratory comparisons of the quality of platelet concentrates. Quantitative lactate and lactate production rate can be used to evaluate the quality of platelets concentrates during storage. However, the formation of lactate depends on the platelet count and the amount of glucose in the product making the comparison more difficult.

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor.

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.

Establishment of immunoassay for platelet-derived soluble glycoprotein VI, a novel platelet marker.

Soluble Glycoprotein VI (GPVI) is an attractive marker for disorders marked by platelet activation, such as thrombotic microangiopathy, myocardial infarction, and stroke. Several groups have already developed an immunoassay for soluble GPVI; however, there are several discrepancies between the groups' assays. In this study, we prepared the two types of recombinant soluble GPVI, the monomeric form GPVI (GPVI-His) and the dimeric form of GPVI (GPVI-Fc), moreover, we generated four anti-GPVI antibodies, F1232-7-1 (7S1), F1232-10-2 (10S2), F1232-19-1 (19D1), and F1232-21-1 (21D1). The former 2 antibodies (7S1 and 10S2) had a high affinity for both GPVI-His and GPVI-Fc, while the latter 2 antibodies (19D1 and 21D1) showed a high affinity for GPVI-Fc but low affinity for GPVI-His. All of the antibodies comparably recognized surface GPVI on resting platelets. Furthermore, we established two immunoassays for soluble GPVI, 7S1/10S2-HRP and 19D1/21D1-HRP (capture antibody/detection antibody). 7S1/10S2-HRP showed equivalent reactivity with GPVI-His and GPVI-Fc, whereas 19D1/21D1-HRP had high affinity for GPVI-Fc but low reactivity with GPVI-His. In terms of reactivity with platelet-derived soluble GPVI, 7S1/10S2-HRP demonstrated sensitive detection whereas 19D1/21D1-HRP was nonreactive. Taken together, 7S1/10S2-HRP is a suitable candidate for a reliable soluble GPVI immunoassay as it has a high affinity for monomeric GPVI.

An improved flow cytometric immunobead array to detect autoantibodies in plasma from patients with immune thrombocytopenic purpura.

BACKGROUND: Autoantibodies against platelet glycoproteins (GPs) play an important role in immune thrombocytopenic purpura (ITP). This study was to develop an improved flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies in ITP patient plasma. METHODS: Plasma samples were isolated from 71 ITP patients and 136 non-ITP controls and incubated with platelets from normal individuals. After washing, platelets were lysed and the platelet lysates were incubated with polystyrene microbeads coupled with monoclonal antibodies against human GPs IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51). Platelet GP-autoantibody complexes were detected by flow cytometry using a FITC-labeled secondary antibody. RESULT: Autoantibodies against platelet GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in plasma from ITP patients, as indicated by high mean fluorescent intensity values when microbeads with antibodies SZ1, SZ2, SZ21, SZ22, and SZ51 were used. In ROC analysis, values of the area under the curve were 0.74, 0.83, 0.80, 0.79 and 0.87, respectively. Compared with the previously reported assays, this new FCIA eliminated the need of isolating platelets from ITP patients without compromising assay sensitivity and accuracy in predicting ITP. CONCLUSION: This simplified FICA assay may be more suitable for ITP diagnosis in clinical laboratory settings.

Platelet-activating factor (PAF) receptor expression is associated with histopathological stage and grade and patients' survival in gastric adenocarcinoma.

Platelet activating factor (PAF) has been considered as potent inflammatory lipid mediator that exerts its actions by binding to PAF receptor (PAFR). PAF/PAFR system has been implicated in several pathophysiological states, including tumor progression, angiogenesis and metastasis. The present study aimed to evaluate the clinical significance of PAFR expression in gastric adenocarcinoma. PAFR protein expression was assessed immunohistochemically on 54 gastric adenocarcinoma tissue samples and was analyzed in relation with clinicopathological parameters, tumor proliferative capacity and patients' survival. PAFR was abundantly expressed in all gastric adenocarcinoma cases examined. Increased PAFR expression was significantly more frequently observed in well/moderately compared to poorly differentiated gastric adenocarcinoma cases (p=0.011). PAFR expression was significantly increased in intestinal- compared to diffuse-type cases (p=0.020). Elevated PAFR expression was significantly associated with smaller tumor size, absence of lymph node and organ metastasis and low tumor histopathological stage (p=0.025, p<0.001, p=0.009 and p<0.001, respectively). Additionally, patients presenting elevated PAFR expression had significantly longer survival times compared to those with low PAFR expression (log-rank test, p<0.001). These results support an important potential role of PAFR signalling in gastric malignant disease progression and render further research in this field a necessity.

Proteomic analysis of platelet N-glycoproteins in PMM2-CDG patients.

UNLABELLED: PMM2-CDG, the most frequent congenital disorder of N-glycosylation, is an autosomal recessive disease with a multisystem presentation. PMM2-CDG patients show an increased risk for thrombosis, which might be in part due to spontaneous platelet aggregations as previously described. A potential hypoglycosylation of platelet proteins in these patients might explain this increased reactivity, as removal of sialic acid from platelets, particularly of GPIbα, leads to enhance platelet aggregation and clearance from the circulation. This study is the first one that has evaluated the glycosylation status of platelet proteins in 6 PMM2-CDG patients using different approaches including immunoblot, RCA120 lectin binding to platelets and expression of different membrane platelet N-glycoproteins by flow cytometry, as well as by platelet N-glycoproteome analysis. RCA120 lectin binding to the platelet membrane of PMM2-CDG patients showed evidence for decreased sialic acid content. However, immunoblot and flow cytometric analysis of different platelet N-glycoproteins, together with the more sensitive 2D-DIGE analysis, suggest that platelet N-glycoproteins, including GPIbα, seem to be neither quantitatively nor qualitatively significantly affected. The increased binding of RCA120 lectin could be explained by the abnormal glycosylation of hepatic proteins being attached to the platelets. CONCLUSIONS: This is the first study that has evaluated the platelet N-glycoproteome. Our findings suggest that platelet proteins are not significantly affected in PMM2-CDG patients. Further studies are still warranted to unravel the mechanism(s) that increase(s) the risk of thrombosis in these patients.