Platelet Factor 4 Levels Research Articles

Suppressive effect of CORM-2 on platelet α-granule exocytosis in sepsis via SNARE/Munc18b complex formation

To investigate the suppressive effect of carbon monoxide-releasing molecule II (CORM-2) on LPS induced platelet α-granule exocytosis in sepsis via soluble N-ethylmaleimide-sensitive factor attached protein receptor/mammalian uncoordinated 18b (SNARE/Munc18b) complex formation. Blood was collected from healthy volunteers' cubital vein, then platelets were isolated by differential centrifugation. Platelets were randomly divided into 5 groups. The control group did not undergo any treatment, the LPS group received 10 mg/L LPS simulation, the CORM-2 group and iCORM-2 group underwent LPS simulation and immediate administration of CORM-2 (10 μmol/L and 50 μmol/L) or iCORM-2 (50 μmol/L), respectively. Samples were incubated in a CO2-incubator at 37 °C, 95% humidity, and 5% CO2. Platelet α-granule contents were detected by using standard enzyme linked immunosorbent assay (ELISA), including platelet factor 4 (PF4), platelet derived growth factor-BB (PDGF-BB), and matrix metalloproteinase-2 (MMP-2). The expression of P-selectin was detected by flow cytometer. Transmission electron microscope and immunofluorescence microscope was used to assess platelet α-granules distribution. Expressions of Munc18b and SNARE proteins including vesicle-associated membrane protein-8 (VAMP-8), synaptosomal-associated protein-23 (SNAP-23) and syntaxin-11 (STX-11) were detected by Western Bolt. The SNARE/Munc18b complex formation was detected by immunoprecipitation. Compared with the control group, levels of PF4, PDGF-BB, MMP-2 and P-selectin in LPS induced platelets were found to markedly elevated, while CORM-2 (10 μmol/L and 50 μmol/L) could decrease platelet α-granule contents exocytosis: [PF4 (μg/L): 7.69±0.58, 6.03±0.71 vs. 10.13±0.82; PDGF-BB (μg/L): 112.71±1.79, 102.91±5.86 vs. 128.78±1.39; MMP-2 (ng/L): 32.94±2.73, 27.58±3.36 vs. 53.26±1.21; P-selectin: (17.14±0.57)%, (15.35±0.68)% vs. (23.78±0.62)%; all P < 0.01]. Transmission electron microscope and immunofluorescence microscope showed that the extent of platelet α-granules assembled to platelet plasma membrane was significantly decreased following CORM-2 treatment. Compared with the control group, the expressions of Munc18b and SNARE proteins and SNARE/Munc18b complex formation in LPS-stimulated platelets were significantly increased, while CORM-2 (10 μmol/L and 50 μmol/L) inhibited these elevations (Munc18b/GAPDH: 0.80±0.08, 0.69±0.01 vs. 0.99±0.09; VAMP-8/GAPDH: 0.72±0.09, 0.50±0.12 vs. 1.18±0.14; SNAP-23/GAPDH: 1.18±0.22, 0.63±0.10 vs. 1.90±0.08; STX-11/GAPDH: 0.76±0.02, 0.57±0.08 vs. 1.16±0.23; VAMP-8/Munc18b: 0.65±0.09, 0.53±0.07 vs. 1.21±0.20; SNAP-23/Munc18b: 0.85±0.07, 0.55±0.09 vs. 1.26±0.08; STX-11/Munc18b: 0.78±0.05, 0.61±0.10 vs. 1.39±0.16; all P < 0.01). Above all, the data showed a dose dependent change. We could suggest that CORM-2 suppressed α-granule exocytosis in LPS-stimulated platelets and the potential mechanisms might involve SNARE/Munc18b complex formation.

Reduced induction of anti-PF4/heparin antibody in RA patients after total knee arthroplasty.

BackgroundHeparin-induced thrombocytopenia is caused by antibodies (Abs) specific to platelet factor 4 (PF4)/heparin complexes. In this study, we evaluated the rates of seroconversion of anti-PF4/heparin Ab between patients with rheumatoid arthritis (RA) and with osteoarthritis (OA) who underwent total knee arthroplasty.MethodsThe subjects of this randomized controlled trial were 124 patients who underwent total knee arthroplasty (TKA) and received edoxaban with or without a foot pump as thromboprophylaxis. We measured anti-PF4/heparin Abs before and 10 days after surgery, as well as preoperative PF4, using commercially available ELISAs. We also used the database of J-PSVT, a hospital-based, prospective cohort study designed to document the effectiveness of thromboprophylactic agents during arthroplasty.ResultsThe rates of seroconversion to anti-PF4/heparin Ab were lower in RA patients (4.0 %) than in OA patients (25.5 %). The anti-PF4/heparin IgG optical density (OD) values did not differ before and after surgery in RA patients. In contrast, there was a significant increase in anti-PF4/heparin IgG OD values in OA patients after TKA. In the J-PSVT data, the postoperative seroconversion rates of anti-PF4/heparin Ab were lower in RA patients (10.4 %) than in OA patients (21.8 %) who received fondaparinux. The titers of anti-CCP Ab were significantly lower in RA patients with postoperative ant-PF4/heparin Ab compared with those without postoperative ant-PF4/heparin Ab There was no significant difference in preoperative PF4 levels between RA patients and OA patients. The heparin-binding affinity of the circulating PF4 was similar between RA patients and OA patients; however, the IgG fractions isolated from the sera of RA patients contained PF4 more frequently (69.2 %) than those from OA patients (10.2 %).ConclusionsOur results showed a reduced likelihood of postoperative anti-PF/heparin Ab production in RA patients compared with OA patients. This suggests that the mechanisms underlying the anti-PF4 immune response in RA patients differ from the mechanisms of the anti-PF4/heparin immune response seen in OA patients after joint replacement.Trial registrationISRCTN 18090286. Registered 8 July 2016

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

BackgroundActivated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma.AimsTo determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity.MethodsPRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin.ResultsPRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone.ConclusionsPEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

Heat therapy promotes the expression of angiogenic regulators in human skeletal muscle.

Heat therapy has been shown to promote capillary growth in skeletal muscle and in the heart in several animal models, but the effects of this therapy on angiogenic signaling in humans are unknown. We evaluated the acute effect of lower body heating (LBH) and unilateral thigh heating (TH) on the expression of angiogenic regulators and heat shock proteins (HSPs) in healthy young individuals. Exposure to LBH (n = 18) increased core temperature (Tc) from 36.9 ± 0.1 to 37.4 ± 0.1°C (P < 0.01) and average leg skin temperature (Tleg) from 33.1 ± 0.1 to 39.6 ± 0.1°C (P < 0.01), but did not alter the levels of circulating angiogenic cytokines and bone marrow-derived proangiogenic cells (CD34(+)CD133(+)). In skeletal muscle, the change in mRNA expression from baseline of vascular endothelial growth factor (VEGF), angiopoietin 2 (ANGPT2), chemokines CCL2 and CX3CL1, platelet factor-4 (PF4), and several members of the HSP family was higher 30 min after the intervention in the individuals exposed to LBH (n = 11) compared with the control group (n = 12). LBH also reduced the expression of transcription factor FOXO1 (P = 0.03). Exposure to TH (n = 14) increased Tleg from 32.8 ± 0.2 to 40.3 ± 0.1°C (P < 0.05) but Tc remained unaltered (36.8 ± 0.1°C at baseline and 36.9 ± 0.1°C at 90 min). This intervention upregulated the expression of VEGF, ANGPT1, ANGPT2, CCL2, and HSPs in skeletal muscle but did not affect the levels of CX3CL1, FOXO-1, and PF4. These findings suggest that both LBH and TH increase the expression of factors associated with capillary growth in human skeletal muscle.

Platelet factor 4 is produced by subsets of myeloid cells in premetastatic lung and inhibits tumor metastasis.

Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor–promoting microenvironment. However, subsets of myeloid cells have also been reported to have anti-tumor properties. It is not clear whether there is a transition between anti- and pro- tumor function of these myeloid cells, and if so, what are the underlying molecular mechanisms. Here we report platelet factor 4 (PF4), or CXCL4, but not the other family members CXCL9, 10, and 11, was produced at higher levels in the normal lung and early stage premetastatic lungs but decreased in later stage lungs. PF4 was mostly produced by Ly6G+CD11b+ myeloid cell subset. Although the number of Ly6G+CD11b+ cells was increased in the premetastatic lungs, the expression level of PF4 in these cells was decreased during the metastatic progression. Deletion of PF4 (PF4 knockout or KO mice) led an increased metastasis suggesting an inhibitory function of PF4. There were two underlying mechanisms: decreased blood vessel integrity in the premetastatic lungs and increased production of hematopoietic stem/progenitor cells (HSCs) and myeloid derived suppressor cells (MDSCs) in tumor-bearing PF4 KO mice. In cancer patients, PF4 expression levels were negatively correlated with tumor stage and positively correlated with patient survival. Our studies suggest that PF4 is a critical anti-tumor factor in the premetastatic site. Our finding of PF4 function in the tumor host provides new insight to the mechanistic understanding of tumor metastasis.

Serum Biomarkers of Allergic Contact Dermatitis: A Pilot Study

Background: Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by repeated skin exposure to contact allergens. The goal of this pilot study was to identify inflammatory proteins which can serve as biomarkers for ACD. Methods: We measured levels of 102 cytokines, chemokines, and growth factors in the sera of 16 ACD patients during acute and remission phases, and 16 healthy volunteers. Results: Serum levels of adiponectin, chemokine (C-C motif) ligand 5 (CCL5), C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), complement factor D (CFD), endoglin, lipocalin-2, osteopontin, retinol-binding protein 4 (RBP4), and platelet factor 4 (PF4) were significantly higher, whereas levels of trefoil factor 3 (TFF3) were significantly lower, in ACD patients than in healthy controls. In ACD patients, serum levels of CCL5 were elevated, whereas levels of TFF3, soluble intercellular adhesion molecule-1 (sICAM-1), and platelet-derived growth factor (PDGF)-AB/BB were found to be lower during the remission phase of the disease. Conclusions: Serum levels of adiponectin, CCL5, CRP, CHI3L1, CFD, endoglin, lipocalin-2, osteopontin, RBP4, PF4, and TFF3 might be exploited as biomarkers for ACD, whereas levels of CCL5, TFF3, sICAM-1, and PDGF-AB/BB might be exploited for evaluation of disease progression and efficacy of ACD treatment.

Investigation of the inflammatory biomarkers of metabolic syndrome in adolescents.

Metabolic syndrome (MetS) is a chronic and multifactorial syndrome characterized by a low-grade chronic inflammation, and a major risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). In our study, we aimed to investigate the serum levels of high sensitive C-reactive protein (hs-CRP), haptoglobin (Hp), α2-macroglobulin (α2-MG), platelet factor-4 (PF-4), fetuin-A, serum amyloid P (SAP) and α1-acid glycoprotein (AGP) in an adolescent population with MetS. This study was performed in 43 (18 males, 25 females) MetS adolescents between the ages of 13 and 17 years (14.70±1.15) and 43 lean controls were matched for age and sex. The serum levels of Hp, α2-MG, PF-4, fetuin-A, SAP and AGP were measured by using a multi-ELISA technique. Serum Hp, fetuin-A (p<0.01) and PF-4, hs-CRP, SAP, AGP (p<0.001) values of the MetS subjects were significantly higher than those of the controls. No difference was found in serum α2-MG levels between the MetS and control groups (p=0.184). This finding suggests the possibility of using these markers in diagnosis of MetS in adolescents to prevent future complications.

Role of Platelet Activation in the Pathogenesis of Heart Failure in End-Stage Renal Disease Patients

Introduction: Heart failure (HF) is prevalent in patients with End-Stage Renal Disease (ESRD) with an incidence of approximately 40%. ESRD patients exhibit both quantitative and qualitative changes in platelets due to the uremic milleu and / or stress caused by the dialysis filtration membranes. Platelet Factor 4 (PF4) and Platelet Derived Growth Factor (PDGF) are released from the alpha granules of activated platelets and play a role in heparin neutralization and angiogenesis, respectively. The purpose of this study was to determine the interplay of platelets and their released mediators and their relevance to the pathogenesis of HF in ESRD patients.Material and Methods: Under IRB approval, ninety blood samples from maintenance hemodialysis patients at Loyola University outpatient dialysis unit were collected prior to dialysis session and stored at -70°C. Twenty-five male and twenty-five female plasma samples from healthy individuals were obtained as a control (George King Biomedical Overland Park, KS). Maintenance hemodialysis patients' and healthy volunteers' plasma samples were used to profile PDGF-BB, Heparin anti Xa, and PF4 using commercial sandwich and competitive ELISA kits (R&D Systems, Minneapolis, MN | Hyphen Biomed, Neuville-sur-oise, France). In addition, patients' HF diagnoses, comorbidities, medications, and clinical laboratory parameters were reviewed through the patients' medical records.Results: Plasma biomarkers PF4 (P < 0.0001; % change = 247.76) and heparin level as measured by an anti Xa methods were significantly increased (P < 0.05) in patients with ESRD in comparison to the values obtained in normal healthy volunteers. PDGF-BB was not found to be significantly increased (P = 0.4045 ; % change = 40.3%). PF4, Heparin levels, and PDGF-BB were not found to be significantly elevated in ESRD (+) HF vs ESRD (-) HF (P > 0). In male ESRD (+) CHF vs ESRD (-) HF, platelet counts were significantly decreased (P = 0.0476 ; % change = -16.5). Systolic BP and PLT count had a positive correlation in both male and female ESRD patients and in male ESRD (+) HF patients (P = 0.016 and P = 0.044, respectively), but not in female ESRD HF (+) patients (P = 0.172). PLT count and PDGF were found to have a positive correlation in ESRD HF (+) patients (P = 0.045). In ESRD (+) HF patients, PF4 had a positive correlation with proBNP (P = 0.045) and Heparin levels had a negative correlation with proBNP (P = 0.045).Discussion: Elevated PF4 in the ESRD patients compared to normals suggest that there may be increased platelet granule release or an upregulation of PF4. The platelets may be activated due to dialysis procedure stress from the previous session, the uremic environment, or heparin-PF4 complexed with antibodies binding to the platelet surface. Elevated heparin levels in the ESRD patients compared to normal suggests that heparinization at dialysis session may lead to an anticoagulated state between sessions in several of these patients. Since these patients are continually heparinized, it is expected to see increased levels of platelet release products such as PF4 due to their mobilization from platelets. Activated platelets may lead to platelet exhaustion and thrombocytopenic responses. Interestingly thrombocytopenia is only noticed in male ESRD (+) HF patients suggesting gender differences and the potential role of hormonal regulation. These results suggest that both the quantitative and qualitative defects in platelets play an important role in the mediation of the pathogenesis of HF in ESRD patients underscoring the potential benefit of antiplatelet drugs. DisclosuresNo relevant conflicts of interest to declare.

Platelet Activation Markers Are Associated with Crohn's Disease Activity in Patients with Low C-Reactive Protein.

In assessing Crohn's disease (CD) activity, C-reactive protein (CRP) is an important indicator of inflammation; however, it is not necessarily associated with the Crohn's Disease Activity Index (CDAI), particularly in patients with low CRP. Recently, platelet activation factors have been recognized due to their importance in the inflammatory response. In this study, we examined associations between the CDAI and platelet factor 4 (PF-4), β-thromboglobulin (β-TG), and other coagulation and fibrinolysis factors. We aimed to find a new marker for evaluating disease activity in patients with CD and low CRP. Nine markers, including CRP, platelet count, white blood cell count, fibrin and fibrinogen degradation product, fibrinogen, thrombin-antithrombin complex, prothrombin fragments 1 + 2, PF-4, and β-TG were evaluated in 47 patients with CD and low CRP (<1.0 mg/dl). Patients were assigned to high or low disease activity groups, CDAI-H (CDAI ≥ 150) and CDAI-L (CDAI < 150), respectively. CDAI-H exhibited significantly higher PF-4 and β-TG levels than CDAI-L (P < 0.01). Other markers were not significantly different between groups. CDAI was positively correlated with the levels of PF-4 and β-TG (P = 0.0033 and 0.0024; r = 0.4202 and 0.4321, respectively). Receiver operating characteristic curve analyses of PF-4 and β-TG showed high sensitivity (61.9 and 81%, respectively) and specificity (84.7 and 69.2%, respectively) for diagnosing active CD. Among eight potential markers, PF-4 and β-TG were the most highly correlated with CDAI in patients with CD and low CRP. PF-4 and β-TG levels showed promise as new markers for assessing CD in patients with low CRP.

Serum biomarkers in uncontrolled no heart-beating donors may identify kidneys that will never work after transplantation.

Kidneys from uncontrolled non heart-beating donors achieve a good level of renal function after transplantation. However, a number of them will never function in the recipient. Our aim was to determine if serum biomarkers associated with platelet activity, inflammation and the nitric oxide system in uncontrolled non heart-beating donors may help to predict no renal function recovery after renal transplantation. Serum levels of interleukin (IL)-6, IL-10, intercellular cell adhesion molecule-1 (ICAM-1), cyclic guanosine monophosphate (cGMP), nitrite+nitrate and platelet factor-4 (PF4) were measured using enzyme-linked immunosorbent assay (ELISA) kits in 88 uncontrolled non heart-beating donors divided according to the renal functionality achieved in the recipients into functional (n=76) and non functional (n=12). Kidneys from donors with higher IL-6 levels (>900pg/ml) were functional after transplantation. Serum cGMP levels below 372.3 fmol/l were also associated with kidneys that recovered the renal function. However, serum levels of PF4 showed the best correlation with recovery of renal functional in the recipients since they were significantly lower in the donors whose kidneys functioned after transplantation. Serum PF4 levels in uncontrolled non heart-beating donors may be a good predictor for kidneys that never will reach functional recovery. Some serum cGMP, IL-6 and IL-10 levels may simply help identify kidneys that will function after transplantation.

Circulating TGF-β1 and VEGF and risk of cancer among liver transplant recipients.

Transplant recipients have elevated cancer risk, perhaps partly due to direct carcinogenic effects of immunosuppressive medications. Experimental evidence indicates that calcineurin inhibitors given to transplant recipients increase cellular expression of transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF), which could promote cancer. To assess the potential role of these pathways in the transplantation setting, we conducted a case–control study nested in a cohort of liver recipients. Cases had nonmelanoma skin cancer (N = 84), cancer of the lung (N = 29), kidney (N = 20), or colorectum (N = 17), or melanoma (N = 3). We selected N = 463 recipients without cancer as controls. TGF-β1 and VEGF levels were measured in sera obtained, on average, approximately 3 years before case diagnosis/control selection. We also measured platelet factor 4 (PF4), a marker of ex vivo platelet degranulation, because TGF-β1 and VEGF can be released from platelets, and we developed a statistical model to isolate the platelet-derived fraction from the remaining circulating component. Compared with controls, lung cancer cases had higher levels of TGF-β1 (median 22.8 vs. 19.4 ng/mL, P = 0.02) and VEGF (277 vs. 186 pg/mL, P = 0.02). However, lung cancer cases also had higher platelet counts (P = 0.08) and PF4 levels (P = 0.02), while residual serum levels of TGF-β1 and VEGF, after accounting for PF4, were unassociated with lung cancer (P = 0.40 and P = 0.15, respectively). Associations were not seen for other cancers. In conclusion, TGF-β1 and VEGF levels were increased in association with lung cancer among transplant recipients, which may be explained by increased platelet counts and platelet degranulation in lung cancer cases.

Mechanism of action of antiplatelet drugs on decompression sickness in rats: a protective effect of anti-GPIIbIIIa therapy.

Literature highlights the involvement of disseminated thrombosis in the pathophysiology of decompression sickness (DCS). We examined the effect of several antithrombotic treatments targeting various pathways on DCS outcome: acetyl salicylate, prasugrel, abciximab, and enoxaparin. Rats were randomly assigned to six groups. Groups 1 and 2 were a control nondiving group (C; n = 10) and a control diving group (CD; n = 30). Animals in Groups 3 to 6 were treated before hyperbaric exposure (HBE) with either prasugrel (n = 10), acetyl salicylate (n = 10), enoxaparin (n = 10), or abciximab (n = 10). Blood samples were taken for platelet factor 4 (PF4), thiobarbituric acid reactive substances (TBARS), and von Willebrand factor analysis. Onset of DCS symptoms and death were recorded during a 60-min observation period after HBE. Although we observed fewer outcomes of DCS in all treated groups compared with the CD, statistical significance was reached in abciximab only (20% vs. 73%, respectively, P = 0.007). We also observed significantly higher levels of plasmatic PF4 in abciximab (8.14 ± 1.40 ng/ml; P = 0.004) and enoxaparin groups (8.01 ± 0.80 ng/ml; P = 0.021) compared with the C group (6.45 ± 1.90 ng/ml) but not CD group (8.14 ± 1.40 ng/ml). Plasmatic levels of TBARS were significantly higher in the CD group than the C group (49.04 ± 11.20 μM vs. 34.44 ± 5.70 μM, P = 0.002). This effect was prevented by all treatments. Our results suggest that abciximab pretreatment, a powerful glycoprotein IIb/IIIa receptor antagonist, has a strong protective effect on decompression risk by significantly improving DCS outcome. Besides its powerful inhibitory action on platelet aggregation, we suggest that abciximab could also act through its effects on vascular function, oxidative stress, and/or inflammation.

Elevated Release of Beta-thromboglobulin and Platelet Factor 4 in Cerebral Infarction Patients with Branch Atheromatous Disease: A Preliminary Report.

This study evaluated the levels of the platelet activation markers beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) in patients with branch atheromatous disease (BAD). Patients with newly diagnosed cerebral infarctions were recruited into the study; those with cardiogenic cerebral infarctions were excluded. Beta-TG and PF4 levels were measured before therapeutic intervention and compared between patients with and without BAD; Welch’s t-test was used to determine significant differences between the groups. A total of 15 subjects were enrolled in the study, and 8 were diagnosed with BAD. Beta-TG (P = 0.031) and PF4 (P = 0.041) levels were significantly higher in the BAD patients than in the non-BAD patients. Platelet activity is normally elevated in patients with cerebral infarctions, but is elevated to an even greater extent in BAD patients. The evaluation of beta-TG and PF4 levels may be beneficial for the elucidation of BAD.

Serum Platelet Factor 4 Is a Promising Therapeutic Response and Prognostic Marker in Patients with Newly Diagnosed Multiple Myeloma

Background: Angiogenesis is closely related with progression and prognosis of multiple myeloma (MM). Imbalance between pro- and anti-angiogenic factors leads to aberrant angiogenesis in MM. Platelet factor 4(PF4) is a potent antiangiogenic factor. It has been shown that PF4 and its p17-70 peptide can inhibit myeloma proliferation and angiogenesis both in vitro and in vivo. In addition, PF4 also directly induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in MM. We identified the peptide (m/z7763.24) with decreased relative intensity in newly diagnosed MM as PF4. Downregulation of PF4 in MM was revealed in several previous researches. However, the predictive analysis of serum PF4 level in newly diagnosed MM has not been well elucidated. Thus in the current study, we proposed to assess if serum PF4 could be a therapeutic response and prognostic marker in patients with newly diagnosed MM.Materials and methods: 62 newly diagnosed MM patients (35 male: 27 female) with a median age of 56.5 years (range 44-73) were recruited. The median follow-up duration was 21 months (range 6–42 months). After two courses of chemotherapy and oral thalidomide, 30 patients failed to achieve complete remission and very good partial remission (non-CR&VGPR) and 30 cases gained CR&VGPR. The relative intensity of PF4 was compared among different MM patients and healthy controls. Results were validated by western blot. Sera of 62 MM patients were gained pre- and post-treatment. Sera from 60 healthy donors were used as controls. Serum PF4 was quantified by ELISA. Independent sample t-test and linear regression were employed to do statistic analyses. Kaplan-Meier method was employed for survival analysis. Log rank test was used for significance analysis. Multivariate analysis of overall survival (OS) used Cox-regression.Results: The relative intensity of PF4 were significantly decreased in newly diagnosed and non-CR&VGPR MM patients, comparing with CR&VGPR patients and healthy controls(p<0.05). Weak PF4 immunoreactive bands were seen in newly diagnosed and non-CR&VGPR MM cases. ELISA demonstrated that mean serum PF4 concentrations in the newly diagnosed group (0.6676±0.2755μg/L) and non-CR&VGPR group (0.6294±0.2732μg/L) significantly differed from the healthy control group (2.4329±0.9709μg/L) and CR&VGPR group (2.4179±0.9605μg/L). Linear regression analysis showed no correlation between PF4 content and platelet count. The PF4 serum concentration was lower in patients with elevated β2-microglobulin (p<0.001), advanced ISS (p=0.003), p53 mutation (p=0.001) and higher level of creatinine (p<0.001). Newly diagnosed MM patients with higher PF4 serum concentration were prone to achieve CR&VGPR (p<0.001).We did not identify any significant distinction in PF4 serum concentration when grouping the patients by age (p=0.988), lactate dehydrogenase (p=0.755), albumin (p=0.305) or hemoglobin (p=0.962). The median survival time of newly diagnosed MM patients in lower serum concentration group (<median serum concentration) was 20.00 months (95%CI: 16.84, 23.16), while in higher serum concentration group (≥median serum concentration) 28.00 months (95%CI: 26.37, 29.63). Kaplan–Meier analyses of OS showed that patients with higher serum concentration of PF4 had a significantly superior outcome and lower serum concentration of PF4 was associated with an unfavorable OS (14.1±8.3% versus 23±8.2%, P=0.001). In univariate survival analysis, the presence of β2-microglobulin, p53 mutation, treatment response and creatinine were also significant predictors of survival, whereas survival was independent of gender, age, lactate dehydrogenase, ISS, albumin, hemoglobin. In multivariate analysis with a Cox regression model, the independent variables associated with a poor OS were p53 mutation (p=0.0296), β2-microglobulin (p=0.0393), treatment response(p=0.031). In addition to these known prognostic factors, MM patients with the low serum PF4 concentration had a significantly inferior outcome (p=0.006).Conclusion: We speculatethatserum PF4 is a promising therapeutic response and prognostic marker in patients with newly diagnosed MM. DisclosuresNo relevant conflicts of interest to declare.

Levels of angiogenic proteins in plasma and platelets are not different between patients with hepatitis B/C-related cirrhosis and patients with cirrhosis and hepatocellular carcinoma

Increasing evidence suggests that levels of angiogenic proteins within blood platelets change at the earliest stages of cancer development and may thus provide a promising diagnostic and prognostic tool. Patients with cirrhosis have increased risk of developing hepatocellular carcinoma (HCC). We aimed to study whether development of HCC in hepatitis-related cirrhosis results in changes in platelet levels of angiogenic proteins. We studied the intraplatelet levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), endostatin, platelet factor 4 (PF4) and thrombospondin type 1 (TSP-1) in 38 consecutive patients with hepatitis B- or C-related liver cirrhosis with or without HCC in addition to plasma levels of the same proteins. Twenty healthy volunteers were included to establish reference values for the various tests. Intraplatelet levels of VEGF, bFGF, HGF and endostatin were significantly higher in patients compared to controls. Intraplatelet levels of PDGF, PF4 and TSP-1 were comparable between patients and controls. Plasma levels of VEGF, bFGF and endostatin were comparable between patients and controls. Plasma levels of PDGF, PF4 and TSP-1 were decreased in patients, but this difference disappeared when levels were corrected for platelet count. Intraplatelet and plasma levels of all proteins assessed were comparable between patients with and without HCC. In conclusion, the intraplatelet levels of some angiogenic proteins are elevated in cirrhosis, but do not discriminate between patients with and without HCC. Thus, intraplatelet levels of angiogenic proteins do not seem useful as diagnostic or prognostic biomarker of HCC in cirrhotic patients.

THROMBOPHILIA AND INTENSITY OF THE INTRAVASCULAR MICROCOAGULATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE, ATHEROTHROMBOSIS

Aim. To study the intensity of the intravascular microcoagulation in patients with atherothrombosis (AT), chronic obstructive pulmonary disease (COPD) and thrombophilia. Material and methods. 115 patients were enrolled into the study: 37 patients with COPD (22 with thrombophilia and 15 without thrombophilia); 37 patients with AT (23 with thrombophilia and 14 without thrombophilia); 41 patients with COPD+AT (24 with thrombophilia and 17 without thrombophilia). In the control group 53 healthy people (39 with thrombophilia and 14 without thrombophilia) were included. Median age was 62 (56.5, 69) years. The intensity of intravascular microcoagulation was measured: platelet factor 4 (PF4) by ELISA, D-dimer by ELISA, XIIa-dependent fibrinolysis (time). Polymerase chain reaction was used for detection of such trombophilia parameters as Leiden mutation, prothrombin gene mutation 20210A, mutation in the gene of methylenetetrahydrofolate reductase (MTHFR) and plasminogen activator inhibitor-1 (PAI-1). Results. The level of PF4 and D-dimer was significantly higher while fibrinolytic activity was lower in COPD (103.5 ME/ml, 70 ng/ml, 650 s, respectively), AT (127.1 ME/ml, 96,9 ng/ml, 850 s, respectively) and COPD+AT (136.5 ME/ml, 106.5 ng/ml, 1148 s, respectively) groups as compared with the control group (p&lt;0.05). When analyzing the level of intravascular coagulation parameters in patients with thrombophilia and without it, levels of PF4, D-dimer and XIIa-dependent fibrinolysis (time) were significantly higher in thrombophilia subgroups among all patients (p&lt;0.05). In patients with thrombophilia levels of these parameters were significantly higher in those with two or more thrombophilia gene polymorphisms than with one (p&lt;0.01). Conclusion. The intensity of intravascular microcoagulation increases in patients with thrombophilia, atherothrombosis and COPD.

Expression of angiogenesis regulatory proteins and epithelial-mesenchymal transition factors in platelets of the breast cancer patients.

Platelets play a role in tumor angiogenesis and growth and are the main transporters of several angiogenesis regulators. Here, we aimed to determine the levels of angiogenesis regulators and epithelial-mesenchymal transition factors sequestered by circulating platelets in breast cancer patients and age-matched healthy controls. Platelet pellets (PP) and platelet-poor plasma (PPP) were collected by routine protocols. Vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), thrombospondin-1 (TSP-1), platelet factor 4 (PF4), and transforming growth factor-β1 (TGF-β1) were measured by enzyme-linked immunosorbent assay. Angiogenesis-associated expression of VEGF (2.1 pg/106 platelets versus 0.9 pg/106 platelets, P < 0.001), PF4 (21.2 ng/106 platelets versus 10.2 ng/106 platelets, P < 0.001), PDGF-BB (42.9 pg/106 platelets versus 19.1 pg/106 platelets, P < 0.001), and TGF-β1 (15.3 ng/106 platelets versus 4.3 ng/106 platelets, P < 0.001) differed in the PP samples of cancer and control subjects. In addition, protein concentrations were associated with clinical characteristics (P < 0.05). Circulating platelets in breast cancer sequester higher levels of PF4, VEGF, PDGF-BB, and TGF-β1, suggesting a possible target for early diagnosis. VEGF, PDGF, and TGF-β1 concentrations in platelets may be associated with prognosis.

Identification Of Markers Of Thrombotic Risk and Response To Cytoreductive Intervention In Philadelphia-Chromosome Negative Myeloproliferative Neoplams

BackgroundThrombosis is a major cause of morbidity and mortality in the Philadelphia-chromosome negative myeloproliferative neoplasms (MPN). However, the pathogenesis of thrombosis in patients with MPN is poorly understood. MPNs are associated with elevated levels of inflammatory cytokines and abnormal platelet activation including elevated platelet factor 4 (PF4), B-thromboglobulin (B-TG) and p-selectin levels. However the relationship between these inflammatory markers and the onset of thrombosis is unclear. Current treatment guidelines recommend the use of cytoreductive agents to decrease thrombosis risk based on history of prior thrombosis and age. The effect of cytoreduction on inflammatory markers has only been partially characterized. Furthermore, current clinical guidelines fail to predict thrombotic events in all patients. Thus, more accurate risk stratification is necessary. AimTo determine if levels of p-selectin and PF4 correlate with thrombotic events and if levels of p-selectin and PF4 are affected by cytoreductive therapy. MethodsBiobanked samples from 16 patients with a confirmed diagnosis of chronic phase MPN (per WHO 2008 criteria) were included in this study. Seven patients had primary myelofibrosis, five had polycythemia vera, and four had essential thrombocythemia. Median age of patients was 65.5 (range 44-81). Fourteen patients had a JAK2 V617F mutation. Four patients with samples collected before and after thrombotic episodes, four patients with post-thrombotic samples, four without a history of thrombosis, and four patients with samples obtained before and after cytoreduction were analyzed. Patient characteristics are summarized in Table 1. Plasma samples were analyzed for levels of PF4 and p-selectin using commercially-available ELISA assays under standard conditions. [Display omitted] ResultsMeasurement of p-selectin levels in MPN patients with a history of a thrombotic event versus disease matched control MPN patients with no history of thrombosis demonstrated an increase in the level of p-selectin in patients with thrombosis. Elevated levels of PF4 were noted in only one patient who had a history of thrombosis, and who was maintained on Aspirin. This suggests that elevated levels of p-selectin may be an indicator of thrombosis risk in patients with MPN.Levels of PF4 and p-selectin were next assessed in patients who had suffered a thrombotic event, both before and after the event had occurred. No changes in p-selectin or PF4 levels pre and post thrombosis were noted.The effect of cytoreduction was measured using samples from patients pre- and post-cytoreduction. No differences in the level of PF4 were noted with cytoreduction. In contrast, reductions in p-selectin were noted in two patients in whom cytoreduction was achieved with Ruxolitinib, but not in two patients in whom cytoreduced with hydroxyurea or pomalidomide. This suggesting that Ruxolitinib may have role in thrombosis risk reduction.Platelet levels were recorded for all 16 patients. There was no difference in platelet levels in each group of patients. In contrast to the changes observed with p-selectin, there was no difference in platelet levels in patients with out a thrombotic event compared to those with a history of thrombosis. This suggests that platelet activation, as measured by p-selectin, may be a stronger indicator of thrombotic risk than platelet levels. ConclusionsImprovements in risk stratification of thrombotic risk for patients with MPN are needed. Platelet activation in MPNs is abnormal and may serve as an additional marker of thrombotic risk. Our results suggest that levels of p-selectin, but not PF4, may be associated with increased thrombotic risk. Furthermore, treatment with the JAK1/2 inhibitor Ruxolitinib resulted in reduction in levels of p-selectin, suggesting that the role of Ruxolitinib in preventing thrombotic event should be further investigated. Additional studies with larger numbers are in progress to validate these results. [Display omitted] Disclosures:Rampal:Foundation Medicine: Consultancy.

VEGF Levels in Plasma in Relation to Platelet Activation, Glycemic Control, and Microvascular Complications in Type 1 Diabetes

OBJECTIVEIncreased levels of vascular endothelial growth factor (VEGF) in human plasma samples have suggested that circulating VEGF is a cause of endothelial dysfunction in diabetes mellitus. However, artificial release of VEGF from platelets as a source of VEGF in plasma samples, as also occurs in serum samples, has not been ruled out in these studies.RESEARCH DESIGN AND METHODSWe determined VEGF levels in plasma collected in both citrate and PECT, a medium that inactivates platelets, in a cross-sectional cohort of 21 healthy subjects and 64 patients with type 1 diabetes. In addition, we evaluated whether VEGF levels in both types of plasma correlated with the presence of diabetes, glycemic control, markers of in vivo or ex vivo platelet activation, and degree of diabetic retinopathy and nephropathy.RESULTSVEGF levels were invariably low in PECT plasma of both nondiabetic and diabetic subjects and were unrelated to any other diabetes-related variable studied. In contrast, VEGF levels in citrate plasma were 150% higher in diabetic patients than in control subjects and correlated with diabetes-related variables. Multiple linear regression analysis showed that levels of platelet factor 4, a marker for ex vivo platelet activation, and HbA1c were the independent predictors of VEGF levels in citrate plasma. Platelet activation, in vivo and ex vivo, was similar in diabetic persons and control subjects.CONCLUSIONSLike serum, citrate plasma is not suitable for reliable measurements of circulating VEGF. The low levels of VEGF in vivo, as represented by measurements in PECT plasma in our study, do not support a role of circulating VEGF in endothelial dysfunction in type 1 diabetes. Higher levels of VEGF in citrate plasma samples of diabetic persons do not represent the in vivo situation, but mainly originate from higher artificial ex vivo release from platelets correlating with the degree of glycemic control.

Platelet factor 4 and β-thromboglobulin in blood plasma of patients with acute exacerbation of chronic obstructive pulmonary disease

To explore the role of platelet factor 4 (PF4) and β-thromboglobulin (β-TG) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). A total of 71 AECOPD patients and 50 chronic obstructive pulmonary disease (COPD) patients within a stable stage were admitted into Beijing Chaoyang Hospital from January 2008 to June 2010. And another 40 healthy volunteers were selected as control group. The data of demographics, arterial blood gas analysis and pulmonary function parameters was collected and analyzed. The plasma levels of PF4 and β-TG were measured by enzyme-linked immunosorbent assay (ELISA). Platelet count was measured by hematology analyzer. Statistical analysis was used for PF4, β-TG and platelet count. Spearman rank correlation was used for correlation analysis. No differences in age and gender existed among the AECOPD, stable and control groups. The plasma level of PF4 in the AECOPD group (2.28 µg/L) was significantly higher than that of the stable group (2.01 µg/L) and control group (1.57 µg/L) (both P < 0.05). The level of β-TG in AECOPD was 2.32 µg/L and it was significantly higher than that of the stable group (1.85 µg/L) and control group (1.29 µg/L) (both P < 0.05). The differences in platelet counts were insignificant between the AEC OPD group ((196 ± 67) ×10(9)/L), stable group ((194 ± 50) ×10(9)/L) and control group ((190 ± 48) ×10(9)/L). AECOPD group was divided into moderate, severe and very severe groups by pulmonary function parameters. The levels of PF4 and β-TG in very severe group were significantly higher than those in moderate and severe groups (P < 0.05). A significant positive correlation was observed between PF4 and β-TG (r = 0.518, P < 0.01). The levels of PF4 and β-TG were negatively correlated with FEV1%, FEV1/FVC and PaO2 (all P < 0.05). Abnormal platelet activation exists in AECOPD. And the levels of PF4 and β-TG may reflect the severity of AECOPD and can be used as the markers of estimating prethrombotic state.