Cellular Response to Individual Components of the Platelet Concentrate

Platelet concentrates and especially their further product platelet lysate, are widely used as a replacement for cell culturing. Platelets contain a broad spectrum of growth factors and bioactive molecules that affect cellular fate. However, the cellular response to individual components of the human platelet concentrate is still unclear. The aim of this study was to observe cellular behavior according to the individual components of platelet concentrates. The bioactive molecule content was determined. The cells were supplemented with a medium containing 8% (v/v) of platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The results showed a higher concentration of fibrinogen, albumin, insulin growth factor I (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), in the groups containing plasma. On the other hand, chemokine RANTES and platelet-derived growth factor bb (PDGF-bb), were higher in the groups containing platelet proteins. The groups containing both plasma and plasma proteins showed the most pronounced proliferation and viability of mesenchymal stem cells and fibroblasts. The platelet proteins alone were not sufficient to provide optimal cell growth and viability. A synergic effect of platelet proteins and plasma was observed. The data indicated the importance of plasma in platelet lysate for cell growth.

Platelet Protein-Related Abnormalities in Response to Acute Hypoglycemia in Type 2 Diabetes

IntroductionPatients with severe COVID-19 infections have coagulation abnormalities indicative of a hypercoagulable state, with thromboembolic complications and increased mortality. Platelets are recognized as mediators of inflammation, releasing proinflammatory and prothrombotic factors, and are hyperactivated in COVID-19 infected patients. Activated platelets have also been reported in type 2 diabetes (T2D) patients, putting these patients at higher risk for thromboembolic complications of COVID-19 infection.MethodsA case-control study of T2D (n=33) and control subjects (n=30) who underwent a hyperinsulinemic clamp to induce normoglycemia in T2D subjects: T2D: baseline glucose 7.5 ± 0.3mmol/l (135.1 ± 5.4mg/dl), reduced to 4.5 ± 0.07mmol/l (81 ± 1.2mg/dl) with 1-hour clamp; Controls: maintained at 5.1 ± 0.1mmol/l (91.9 ± 1.8mg/dl). Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement was used to determine a panel of platelet proteins.ResultsProthrombotic platelet proteins were elevated in T2D versus controls: platelet factor 4 (PF4, p<0.05); platelet glycoprotein VI (PGVI p<0.05); P-selectin (p<0.01) and plasminogen activator inhibitor I (PAI-1, p<0.01). In addition, the antithrombotic platelet-related proteins, plasmin (p<0.05) and heparin cofactor II (HCFII, p<0.05), were increased in T2D. Normalization of glucose in the T2D cohort had no effect on platelet protein levels.ConclusionT2D patients have platelet hyperactivation, placing them at higher risk for thromboembolic events. When infected with COVID-19, this risk may be compounded, and their propensity for a more severe COVID-19 disease course increased.Clinical Trial Registrationhttps://clinicaltrials.gov/ct2/show/NCT03102801, identifier NCT03102801.

Accelerated endoplasmic reticulum and coagulation factor processing in platelets are linked with the hypercoagulable state of patients with lung cancer

Background The risk of venous thromboembolism in cancer is nine times higher than in the general population and the second leading cause of death in these patients. Platelets play a key role in tumour growth, metastasis, and cancer-associated thrombosis. Despite this widely observed functional role of platelets in the prothrombotic state of certain cancers, the underlying molecular mechanisms are largely unknown. Methods In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. Cancer-related platelet proteins were statistically characterised by adjusted one-way ANOVA and planned comparisons post hoc tests. Significantly changed platelet proteins were identified by mass spectrometry. Afterward, selected cancer-associated platelet proteins were validated by 1-D and 2-D Western blots and/or functional assessments were made by association analysis with central haemostatic plasma parameters. Results The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins CALR, HSPA5 and P4HB were significantly elevated. Accelerated conversion of the fibrin stabilising factor has been detected in platelets of patients with lung cancer by elevated levels of a F13A1 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma D-dimer levels suggests an enhanced degradation in platelets by the fibrinolytic system. Functional protein association network analysis showed that lung cancer-related protein profiles were involved in platelet degranulation and upregulated ER protein processing. The latter is important in the glycosylation of coagulation factors. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. In addition, several lung cancer-related platelet proteins such as decreased levels of ITGA2B are significantly linked to the plasmatic haemostasis factors D-dimer and fibrinogen. Conclusions These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

Arterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.